Vaccine compositions

ABSTRACT

The present disclosure provides vaccine compositions for prophylaxis and treatment of Zika virus infections comprising Zika virus antigens in immunogenic compositions, and in combination of Zika antigens with one or more arbovirus antigens such as Chikungunya virus and Japanese encephalitis virus antigens, methods of preparation and production of such compositions for use as vaccines for eliciting immune response in mammals against the above mentioned pathogens.

CROSS REFERENCE

This Application is a Continuation of U.S. application Ser. No.15/212,804 filed Jul. 18, 2016, which issued as U.S. Pat. No. 10,588,956on Mar. 17, 2020, and claims priority from Indian Provisional PatentApplication No. 3652/CHE/2015 filed in the Indian Patent Office on Jul.16, 2015.

FIELD OF INVENTION

The present invention discloses vaccine compositions comprising Zikavirus antigens for prophylaxis and treatment of Zika virus infections inmammals. The invention also discloses stable vaccine compositionscomprising Zika virus antigens with one or more arbovirus antigens suchas Chikungunya and/or Japanese encephalitis virus antigens. The presentinvention also relates to the methods of preparation, formulation anduse of the same for simultaneously eliciting immune response to each ofthe above mentioned pathogens in mammals, and suitable for immunizinghuman subjects

BACKGROUND OF THE INVENTION

At present there is no vaccine available in the world for prophylaxis ortreatment against Zika virus infections. Therefore, there is no priorart relevant to the invention disclosed in this application. However,for general understanding of the background and objectives behind thisinvention, the invention is discussed hereinafter in below paragraphs.

The Inventors of this patent application anticipated the epidemicpotential of Zika virus in regions with high prevalence of Aedesmosquitoes, particularly Aedes aegypti that transmits the virus. Theinterest in initiating the Zika vaccine project early on, several monthsbefore the causal link of Zika virus infection to Guillain Bane Syndromeand to microcephaly became public knowledge in December 2015, was thatthere was no preparedness in any country in the world, nor measuresinitiated by anyone at that time to develop a vaccine to stop theongoing virus transmission in countries such as Brazil, and to preventfurther transmission in countries at risk for Zika virus. IncreasedInternational travel to and from regions with ongoing virus transmissionimpose a major risk to initiate an outbreak in countries with highprevalence of Aedes mosquitoes, particularly Ae. aegypti and thosehaving a large naïve population hither to unexposed to the virus. Theclinical picture of Zika virus infection in the early stages withcharacteristic high fever, maculopapular rashes and arthralgia isstrikingly similar to the early onset symptoms of Chikungunya and Denguevirus infections that make differential diagnosis particularlychallenging.

Zika virus vaccine project was initiated at the time when very little orno information was available on virus pathogenesis, genetic diversity,transmission, diagnosis, serological correlates for protection or animalmodels to test the vaccine concepts. From vaccine point of view, therewas no information on whether the virus can be cultured in vitro in cellsubstrates and if yes, which cell substrates are best suitable,mechanism of adaptation to cells, potential virus titers and thefeasibility to manufacture the vaccine product for human administration,as the published information at that time pertained to passaging thevirus in mouse brain which is not suitable for vaccine production.Bharat Biotech initiated steps to start Zika vaccine project in late2014, and commenced the experimental work soon thereafter resulting intothis said patent application.

Arbovirus (arthropod-borne) infections are caused by viruses that arespread by arthropods such as mosquitoes. They cause significant humanillness ranging from mild, asymptomatic infection to acute encephalitisor hemorrhagic fever that can prove fatal. The most significantarboviruses causing human illness belong to three viral families,Togaviridae, Flaviviridae, and Bunyaviridae. Arbovirus infections arerampant in developing countries and cause severe morbidity particularlyin the elderly population. The common characteristic feature ofarbovirus infections caused by Dengue, Chikungunya, Zika, Japaneseencephalitis and West Nile viruses among others is fever, headache,myalgia, joint pains with swelling and maculopapular rashes during theacute phase of the viral infection. Arthralgia is particularly acharacteristic feature of Chikungunya, Dengue and Zika virus fever.Co-infections are common as the arboviruses largely share the samemosquito vectors such as for example Dengue, Chikungunya and Zikaviruses that are transmitted by Aedes mosquitoes. Japanese encephalitisvirus and West Nile viruses are transmitted predominantly by Culexmosquitoes. The problem is acute in developing countries where mosquitovector control programs have been ineffective and largely unsuccessful.The problem is compounded by the fact that there are no robustdiagnostic methods available for diagnosing the disease causing viruseswith certainty. International travel has aided widespread disseminationof these infectious agents, and diseases like Dengue and Chikungunyahitherto confined to tropical countries are now spread geographically tonew areas and to temperate regions. Zika virus is reportedly spread toover 65 countries in the last two years. Autochthonous epidemicoutbreaks reported in few countries in these regions are sustained bythe local population of mosquito vectors.

Zika virus (ZIKV) is an emerging zoonotic arbovirus, belonging to theFlaviviridae family. Like Dengue and Chikungunya viruses, Zika virus canalso be transmitted by Aedes mosquitoes more specifically A. furcifer,A. taylori, A. luteocephalus, A. africanus, A. albopictus andpredominantly by, A. aegyph. Travel tourism to nations where the recentepidemics were reported such as Polynesia has aided the geographicalspread of the virus infection to Brazil, Columbia, Italy and to othercountries. An autochthonous outbreak of the virus was reported in Italycaused by thelocally established Aedes mosquitoes. In Asia, Zika virusinfection has occurred sporadically in Cambodia, Thailand, Indonesia,Malaysia and Bangladesh although large epidemic outbreaks have not beenreported in these regions.

Chikungunya virus (CHIKV) is an Alphavirus of the family Togaviridae.The virus causes self-limiting febrile infection characterized by acuteonset of high fever, headache, myalgia, arthralgia, swelling in jointsand maculopapular rashes. Severe symptoms such hemorrhagia, fulminanthepatitis and neurological symptoms were reported in the more recentepidemics. Chikungunya virus is transmitted by both the Aedes aegyptiand Aedes albopictus mosquitoes. Japanese encephalitis virus (JEV) isalso a flavivirus of the family Flaviviridae and is transmitted largelyby the Culex mosquitoes. JEV is related to Dengue, Yellow fever virus,Zika and West Nile viruses. JEV infection is largely asymptomatic, butin general it causes malaise with fever, headache and other flu-likesymptoms. Rarely, the clinical infection progresses to encephalitis withseizures, spastic paralysis, coma and death. Children are particularlysusceptible. In the countries endemic for JEV, most adults have naturalimmunity after childhood infection. Adults not exposed to the infectionduring childhood are susceptible at any age. The case-fatality rate inJEV caused by encephalitis can be as high as 30%. Neurologicalcomplications or psychiatric sequelae occur in high proportion of thecases with encephalitis. Globally, about 3 billion population is at riskfor JEV infection. A few vaccines for prophylaxis of JEV infection havebeen successfully commercialized. Dengue virus (DENY) is a member ofFlaviviridae family. The arbovirus infections can no longer beconsidered region specific as they are now geographically widespread andare significant public health problem in many parts of the world. Themorbidity caused by the aforementioned arbovirus infections is usuallyhigh, and arthralgia in particular, adversely impacts physical mobilityof the patients. Zika virus causes more serious congenital birthdeformities during infection in pregnancy, and Zika related GuillainBarre syndrome have been confirmed in the ongoing epidemics. Like anyother viral infections, no specific therapeutics is available.Prophylactic vaccination can effectively interrupt Zika virustransmission and a vaccine would be the front line of defense from theZika virus disease.

With this in mind, an effective strategy was developed to preventfurther transmission of Zika virus to protect naïve population incountries with ongoing epdiemics and in countries where active Zikavirus transmission has not been reported as yet. A combination vaccinefor arbovirus infections is good strategy to protect vulnerablepopulation from debilitating illnesses caused by Dengue, Chikungunya,Zika, Japanese encephalitis, West Nile and Yellow Fever viruses.Vaccines for JEV, Yellow Fever and one for Dengue serotypes has beencommercialized and those for West Nile and CHIKV are in clinicaldevelopment. There is no vaccine for Zika virus infection as yet, andthe current invention discloses the methods for development of the firstcandidate Zika vaccines.

However, the choice of antigens to include in such a vaccine kit dependson several factors. The antibody dependent enhancement of virus causedby the Dengue serotypes is well researched and published and so also thecross reactivity of flavivirus antibodies. But what was not clear isthat if there would be such interference or cross reactivity ofprevalent Chikungunya antibodies in the same population that is affectedby Zika virus. Similarly, it was not known if antibodies to Japaneseencephalitis virus could cause antigenic interference in developingimmunity to Zika virus and it was an interesting thought to study thesame. The proposed work provides an insight to any possible immuneinterference caused by prevalent JE and CHIKV antibodies to candidateZika vaccine.

In the current invention, candidate Zika virus vaccines have beendeveloped and tested for potency with various formulations to elicit theappropriate level of immune response to protect against Zika virusdisease. As there was no significant antigenic interference to Zikainduced immune response by Japanese encephalitis virus vaccine andChikungunya virus vaccine when co-administered or combined as acombination vaccine, the formulations were effective in eliciting highlevel of neutralizing antibodies capable of conferring protectionagainst each of the viruses.

OBJECTS OF THE INVENTION

One object of the invention is to provide stable immunogeniccompositions for prophylaxis and treatment of Zika virus infections.

Another object of the invention is to provide methods for adaption andgrowth of Zika virus in Vero cells.

Another object of the invention is to provide methods for thepreparation of inactivated Zika virus vaccine

Another object of the invention is to provide methods for thepurification of Zika virus bulk antigen.

One more object of the invention is to provide methods for Zika virusinactivation by chemical means with formalin, beta propiolactone andhydrogen peroxide

Yet another object of the invention is to provide methods for Zika virusinactivation by physical means such as heat, gamma irradiation andultraviolet light

Yet another object of the invention is to provide methods for thepreparation and formulation of recombinant Zika virus antigenscomprising the prME protein and testing for immunogenicity in animals

A further object of the invention is to provide methods for formulationsof Zika virus antigens with different adjuvants and estimation of immuneresponse to the formulations in animals.

Yet another object of the invention is to provide kinetics of immuneresponse to single dose, two and three doses of formalin and BPLinactivated Zika virus vaccine in animals.

One more object of the invention is to provide immunogenic compositionsfor prophylaxis of Zika and Chikungunya virus infections

Yet another object of the invention is to provide immunogeniccompositions for prophylaxis of Zika and Japanese encephalitis virusinfections

Another object of the invention is to provide immunogenic compositionsfor prophylaxis of Zika, Chikungunya and Japanese encephalitis virusinfections.

SUMMARY OF THE INVENTION

The present invention is directed to compositions and methods ofmanufacture of vaccine formulations for prophylaxis and treatment ofZika virus infections as well as infections caused by other arbovirusessuch as Chikungunya virus and Japanese encephalitis virus.

In one aspect, the invention is directed to vaccine compositions forprophylaxis and treatment of Zika virus infections, wherein the saidcompositions comprise Zika virus antigens in immunogenic compositionsand may also comprise one or more arbovirus antigens such as Chikungunyavirus and Japanese encephalitis virus antigens, along with suitableadjuvants and excipients.

In another aspect, the invention is directed to a method of obtainingthe vaccine formulations by a process which comprises:

-   -   (a) Using Vero cell line as cell substrate for Zika virus        culture    -   (b) Scaling up the Zika virus culture upto a harvest volume of        10 L    -   (c) Inactivating the virus culture either before or after        purification of the virus    -   (d) Purifying the virus culture    -   (e) In another aspect, recombinant cloning and expressing Zika        virus prME protein.

In one embodiment of the invention, Vero cell line was used as the cellsubstrate for culture of Zika virus and was grown in culture medium withor without the use of serum.

In another embodiment of the invention, the Zika virus was adapted byrepeated serial passage in Vero cells to obtain higher titers.

In another embodiment of the invention, Zika virus was passaged in C6/36Ae. albopictus cells followed by growth in Vero cells to increase thetiter.

In another embodiment of the invention, processes for scaling up thevirus culture and further purifying the scaled up virus cultures isdisclosed, wherein the harvest volume was about 8-10 L. The virus waspurified by Capto Core 700 column chromatography and then inactivated.Alternately, the viral harvest was inactivated using various methods.The virus was then purified.

In another embodiment of the invention, inactivation method is selectedfrom a group of Formalin inactivation, Beta Propiolactone (BPL)inactivation, heat inactivation, UV inactivation, gamma inactivation, inthe presence or absence of virus stabilizing agents and amino acids.

In a preferred embodiment of the invention, amino acids were selectedindividually or in combination, from a group L-Histidine, L-Glutamicacid, L-Glycine and L-Aspartic acid and L-Glutamine and human serumalbumin.

In another preferred embodiment of this invention, the purificationmethod is selected by use of cellufine sulphate, DEAE-SephadexCM-sephadex with salt gradient, by gel filtration on Captocore-700,Sepharose CL-4B, ceramic hydroxyapatite column with gradient of 0.2M to0.8M phosphate followed by diafiltration, and ultracentrifugation on a20-60% sucrose gradient, most preferably by Capto Core 700 column.

Another embodiment of the invention is directed to recombinant cloningand expression of Zika virus prME protein is provided. The method ofrecombinant cloning utilizes a site specific transposition of theexpression cassette with the cloned inserts into a baculovirus shuttlevector propagated in E. coli and expressed in insect cells.

In another embodiment of the invention, vaccine formulations areprovided. The vaccine may comprise of one or more arbovirus antigensselected from Zika virus, Chikungunya virus and Japanese encephalitisviruses.

In another embodiment, adjuvants can be selected from a group ofaluminium salts, inulin, algammulin, combination of inulin and aluminiumhydroxide, monophosphoryl lipid A (MPL), resiquimoid, muramyl dipeptide(MDP), N-glycolyl dipeptide (GMDP), poly IC, CpG oligonucleotide,resiquimod, aluminium hydroxide with MPL, any water in oil emulsion, anyoil in water emulsion that contains one or more of the followingconstituents: squalene or its analogues or any pharmaceuticallyacceptable oil, tween-80, sorbitan trioleate, alpha-tocopherol,cholecalciferol or any of the analogues and derivatives of the moleculesthereof, or calcium phosphate or any combination of the adjuvants.

In another embodiment of the invention, the formulations are preparedwith excipients and preservatives.

In another embodiment of the invention, stabilizing agents in thevaccine formulation were used individually or in combinations ofsorbitol, L-glycine, mannitol, L-glutamic acid and human serum albuminin various concentration was used to study the same.

In another embodiment of the invention, the potency of the vaccineformulations have been tested in animal models to show completeprotection from viremia over a wide range of doses.

In another embodiment of the invention, the combination vaccineformulations were also effective in providing adequate protectionagainst Zika, Japanese Encephalitis as well as Chikungunya viruses.

In another embodiment of the invention, the Zika polyclonal antiseraconfers passive immunity in mice against Zika virus infection byprotecting against viremia, while viremia was detected in the controlanimals that persisted up to 6 days after virus challenge.

In another embodiment of the invention, the candidate inactivated Zikavirus vaccine can be administered either as a single dose, or in two ormore doses by intramuscular route

In another embodiment of the invention, assays for neutralizing antibodytiters were conducted to check the antibody levels against vaccineformulations of the present invention which has shown to elicit highlevel of neutralizing antibodies.

In another embodiment of the invention, cross neutralization studiesexhibited that inactivated vaccine formulations of the present inventionwould be equally protective and potent against any Zika virus strain.

In another embodiment of the invention, prME antisera of the presentinvention cross neutralized the MR766 strain of the African genotypeindicating that no serotypes of Zika virus exist.

In another preferred embodiment of the invention, antibody titers toboth BPL inactivated and formalin inactivated Zika vaccine formulationswere higher with aluminium hydroxide than with antigens alone.

In another embodiment of the invention, quality of antibody responses tothe vaccine formulations of the present invention by antibody avidityassays indicated that high affinity antibodies were elicited by thevaccine formulations.

Accordingly, the invention provides a stable vaccine compositioncomprising one or more arbovirus antigens selected from Zika virus,Chikungunya virus and Japanese encephalitis virus, said antigens beingformulated with or without an adjuvant in pharmaceutically acceptablebuffer, wherein the vaccine composition elicits protective immuneresponse to each of the viruses in mammals. The Zika virus antigen ofthe composition is effective for treatment, diagnosis and prophylaxisagainst any genotype/genotypic variants/strains of Zika virus, whereinthe composition is effective against any genotype/genotypicvariants/strains/synthetic Zika viruses that share anywhere between 50%to 100% identity at the amino acid level in any region of the genome.The composition of the invention comprises Zika virus antigens of anygenotype/genotypic variant/strains/synthetic Zika virus, wherein theantibodies against any of the aforementioned Zika virus types crossneutralizes the homologous virus or any heterologous Zika virus strainthat shares at least 50%-100% amino acid identity in any region of itswhole genome, particularly the envelope E protein.

The antigens of Zika virus, Chikungunya virus and Japanese encephalitisvirus of the composition are inactivated whole virion (virus) antigens.Whereas, in another embodiment, the Zika and Chikungunya virus antigensare purified recombinant antigens.

The Zika virus antigen of the invention is prepared using Vero cells ascell substrate by adapting the virus to Vero cells.

The Zika virus antigen of the composition of the invention is a purifiedand concentrated antigen obtained from one or more methods selectedfrom:

-   -   a. ultracentrifugation;    -   b. density gradient centrifugation;    -   c. clarification of the viral harvest using membrane filtration,        followed by purification by column chromatography; and    -   d. tangential flow filtration using membranes with cut off from        100 kDa to 300 kDa, wherein tangential filtration is carried out        either before or after virus inactivation.

Wherein the purification by column chromatography comprises gelfiltration, mixed mode resin column chromatography, ion exchange columnchromatography, affinity matrix chromatography and hydrophobicinteraction chromatography. The column chromatography elutes majority ofthe virus antigen in the flow through such as Capto Core 700, mostpreferably Capto Core 700 wherein the virus sample is purified on CaptoCore 700 column and is eluted in the flow through.

The Zika virus of the composition is inactivated by at least one or moreof a chemical inactivating agent, a physical inactivating agent and anirradiating agent, wherein the inactivation of Zika virus is carried outbefore or after purification of the virus. In an exemplary embodiment,the Zika virus is inactivated by chemical inactivating agent selectedfrom formalin (formaldehyde), beta propiolactone (BPL) and hydrogenperoxide.

In one preferred embodiment the Zika virus is inactivated by any one ofthe following methods selected from:

-   -   a. Formalin treatment at any concentration ranging from 1:500 up        to 1:4000 v/v of formalin:virus, at 8° C. to 37° C., preferably        25±3° C., for at least 1 to 7 days;    -   b. Formalin treatment at any concentration ranging from 1:500 up        to 1:4000 v/v of formalin:virus, at 2° C. to 8° C. for at least        10 to 30 days;    -   c. Beta propiolactone (henceforth BPL) at any concentration        ranging from 1:500 up to 1:4000 v/v of BPL:virus, for at least        24 to 48 hours at temperatures ranging from 8° C. to 30° C.,        preferably 25±3° C., for 48 hours;    -   d. Beta propiolactone at any concentration ranging from 1:500 up        to 1:4000 (BPL:virus, v/v), at 2° C. to 8° C. for at least 3-7        days;    -   e. A combination of BPL and formalin at any of the        aforementioned conditions, preferably BPL inactivation at 1:3000        (BPL:virus, v/v) for 24 hours followed by formalin inactivation        at 1:3000 (formalin:virus, v/v) for 24 to 48 hours at 15° C. to        30° C., preferably 25±3° C.;    -   f. Hydrogen peroxide at any concentration from 0.1 to 3%,        preferably 0.1 to 1% at any temperature from 20-30° C. for 5        minutes to 120 minutes.

In one embodiment, the inactivation of the Zika virus by irradiatingagent comprises inactivation by gamma irradiation by exposure from 20kGy (Kilo Gray) up to 35 kGy, preferably 25 kGy to 30 kGy from a ⁶⁰Cosource.

In another embodiment, the inactivation of the Zika virus by irradiatingagent comprises inactivation by UV irradiation by exposure to 254 nm for30-60 minutes.

In a further embodiment, the virus is inactivated by heat treatment at atemperature between 50° C. to 65° C. for 30 min up to 2 hrs.

The buffer used in the invention may be selected from the listcomprising of phosphate buffer, citrate buffer, phosphate citratebuffer, borate buffer, tris(hydroxymethyl) aminomethane (Tris)containing buffer, succinate buffer, buffers containing glycine orhistidine as one of the buffering agents, wherein phosphate buffer issodium phosphate buffer at concentration of 5 mM up to 200 mM ofphosphate ions of any pH between 6.50 to pH 9, and optionally containingsodium chloride at a concentration of 50 to 200 mM. The buffer maintainsthe pH in a liquid composition above pH 6.5, preferably above pH 7.0throughout the bioprocess from viral culture up to preparation ofpurified inactivated virus bulk.

In one embodiment, the inactivation of Zika virus is carried out in thepresence of a stabilizing agent selected from lactose, sucrose,trehalose, maltose, mannose, iso-maltose, raffinose, stachyose,lactobiose, sorbitol, mannitol, lactobionic acid, dextran, L-glycine,L-histidine, L-glutamic acid, L-aspartic acid and human serum albumin orcombinations thereof. However, in one preferred embodiment, thestabilizing agent may be selected from:

-   -   a. 2% sorbitol and 1% L-glycine;    -   b. 1% sorbitol and 0.5% L-glycine;    -   c. 1% mannitol and 0.5% L-glycine;    -   d. 1% mannitol and 0.5% L-glutamic acid; and    -   e. 1% sorbitol and 0.5% L-glycine, 1% human serum albumin.

In an exemplary embodiment, the inactivation of Zika virus comprisesinactivation of any genotype/strain, live attenuated Zika virus,deactivated virus, virus like particles, chimeric virus particles thatcarry any Zika virus antigens particularly the E protein in anyheterologous virus backbone, in vectored vaccines and infectioussynthetic virus particles derived in vitro or in vivo using the sequenceof any Zika virus genome.

The purified recombinant Zika virus of the invention comprises antigensof Zika virus comprising the envelope (E) protein, membrane (M) proteinexpressed as prME and optionally the non-structural 1 (NS1) protein asvaccine antigens for eliciting immune response for prophylaxis of Zikavirus infections, wherein the Zika virus has the structural proteinsequences as disclosed in SEQ. ID NO:3 and SEQ ID NO:4 corresponding tonucleotide sequences of SEQ ID NO:1 and SEQ ID NO:2 respectively, foruse as vaccine antigens against Zika virus infections caused bygenotypes or variants thereof. The Recombinant DNA constructs comprisesa (i) vector (ii) at least one nucleic acid fragment corresponding toSEQ ID NO:1 or SEQ ID NO:2 encoding the amino acid sequence of theproteins of SEQ ID NO:3, SEQ ID NO:4 respectively which is applicable toany Zika virus protein sequences that share at least 70% amino acididentity to the aforementioned SEQ ID NO:3 and SEQ ID NO:4. Thecomposition of the invention comprises recombinant DNA construct,wherein the vector is an eukaryotic plasmid vector being cloned in aeukaryotic host such as baculovirus for expression in insect cells asvirus like particles (VLPs).

The recombinant protein of Zika virus is obtained by the processcomprising the steps of:

-   -   a. transfecting the recombinant plasmid DNA in insect cells;    -   b. harvesting the cells and isolating the recombinant protein        therefrom;    -   c. purifying the protein by a method selected from ion exchange        chromatography, gel filtration, affinity chromatography,        hydrophobic column chromatography, mixed mode resin        chromatography, diafiltration, ultracentrifugation, density        gradient centrifugation and fractionation with salt.

The structural antigens of Zika virus are expressed in any prokaryoticor eukaryotic expression system including baculovirus mediatedexpression in insect cells.

The vaccine composition of the invention is obtained by a processwherein neutralizing antibodies are largely elicited against theEnvelope protein such as in optimally inactivated virus, live attenuatedvirus, deactivated virus, DNA vaccine, virus like particles, chimericvirus particles that display the Zika virus E protein in anyheterologous virus backbone such as in vectored vaccines and syntheticvirus particles derived from any Zika virus genomic RNA sequence.

The vaccine composition of the invention may further comprise anadjuvant, wherein the adjuvant is selected from the group consisting ofa) aluminum salts comprising aluminum hydroxide, aluminum phosphate,aluminum sulphate phosphate; b) inulin; c) algammulin which is acombination of inulin and aluminum hydroxide; d) monophosphoryl lipid A(MPL); e) resiquimod; f) muramyl dipeptide (MDP); g) N-glycolyldipeptide (GMDP); h) polylC; i) CpG oligonucleotide; j) aluminumhydroxide with MPL; k) any water in oil emulsion; 1) any oil in wateremulsion that contains one or more of the following constituents:squalene or its analogues or any pharmaceutically acceptable oil,tween-80, sorbitantrioleate, alpha-tocopherol, cholecalciferol andaqueous buffer, or any of the analogues and derivatives of the moleculesthereof, wherein one or two or more combination of any of theaforementioned adjuvants when formulated with Zika virus antigenselicits immune response against the virus.

In one preferred embodiment the composition comprises aluminum hydroxidein a concentration range of 0.1 mg to 1.5 mg of aluminum per vaccinedose, preferably 0.25 mg to 0.5 mg aluminum per vaccine dose,

The adjuvant of the composition of the invention confers mucosalimmunity and systemic immunity when administered in mammals.

The vaccine composition with Zika virus antigen is administered at anydose ranging from 0.125 μg to 100 μg per dose with or without anadjuvant, either as a single dose or in two or more doses to elicit animmune response in a mammal.

In one embodiment the invention provides a method of eliciting aprotective immune response in mammals including humans comprisingadministering the vaccine composition of claim 1 by any route comprisingintramuscular, intradermal, subcutaneous, intravenous, oral, intranasalor transcutaneous routes.

The composition of the invention may be administered by any methodcomprising needles and syringes including pre-filled syringes,microneedle patch, needle-free patch, inhalation and nasal sprays.

The invention also provides a method of in vitro or in vivo use of theZika virus antibodies of the composition for preparation ofimmunodiagnostic and immunotherapeutic agents for Zika virus infections.

In one embodiment the vaccine composition comprises Zika virus andJapanese encephalitis virus antigens in a combination vaccine thatelicits protective immune response in mammals against each of theviruses, wherein the Zika virus antigen and Japanese encephalitis virusinactivated antigens are present in the combination vaccine atconcentrations ranging 5 μg to 50 μg of each antigen in apharmaceutically acceptable formulation without an adjuvant, or with anadjuvant.

The adjuvant may be selected from the group consisting of a) aluminumsalts comprising aluminum hydroxide, aluminum phosphate, aluminumsulphate phosphate; b) inulin; c) algammulin which is a combination ofinulin and aluminium hydroxide; d) monophosphoryl lipid A (MPL); e)resiquimod; f) muramyl dipeptide (MDP); g) N-glycolyl dipeptide (GMDP);h) polylC; i) CpG oligonucleotide; j) aluminum hydroxide with MPL; k)any water in oil emulsion; 1) any oil in water emulsion that containsone or more of the following constituents: squalene or its analogues orany pharmaceutically acceptable oil, tween-80, sorbitantrioleate,alpha-tocopherol, cholecalciferol and aqueous buffer, or any of theanalogues and derivatives of the molecules thereof wherein one or two ormore combination of any of the aforementioned adjuvants when formulatedwith Zika virus and Japanese encephalitis virus antigens elicits immuneresponse against the virus. In one preferred embodiment, the adjuvant isaluminum hydroxide with 0.25 mg to 1.0 mg of aluminum content pervaccine dose.

In another embodiment, the vaccine composition comprises Zika virus andChikungunya virus antigens in a combination vaccine that elicitsprotective immune response in mammals against each of the viruses,wherein Zika and Chikungunya virus antigens are present in a combinationvaccine at concentrations ranging from 5 μg to 50 μg of each antigen ina pharmaceutically acceptable formulation without an adjuvant, or withan adjuvant.

The adjuvant may be selected from the group consisting of a) aluminumsalts comprising aluminum hydroxide, aluminum phosphate, aluminumsulphate phosphate; b) inulin; c) algammulin which is a combination ofinulin and aluminium hydroxide; d) monophosphoryl lipid A (MPL); e)resiquimod; f) muramyl dipeptide (MDP); g) N-glycolyl dipeptide (GMDP);h) polylC; i) CpG oligonucleotide; j) aluminum hydroxide with MPL; k)any water in oil emulsion; 1) any oil in water emulsion that containsone or more of the following constituents: squalene or its analogues orany pharmaceutically acceptable oil, tween-80, sorbitantrioleate,alpha-tocopherol, cholecalciferol and aqueous buffer, or any of theanalogues and derivatives of the molecules thereo, wherein two or morecombination of any of the aforementioned adjuvants when formulated withZika virus and Chikungunya virus antigens elicits immune responseagainst the virus. In one preferred embodiment, the adjuvant is aluminumhydroxide at 0.25 mg to 1.5 mg of aluminum content per vaccine dose.

In another embodiment, the vaccine composition comprises Zika virus,Chikungunya virus and Japanese encephalitis virus antigens in acombination vaccine that elicits protective immune response in mammalsagainst each of the viruses, wherein Zika virus, Chikungunya virus andJapanese encephalitis virus antigens are present in a combinationvaccine at concentrations ranging from 5 μg to 50 μg of each antigen ina pharmaceutically acceptable formulation without an adjuvant, or withan adjuvant.

The adjuvant may be selected from the group consisting of a) aluminumsalts comprising aluminum hydroxide, aluminum phosphate, aluminumsulphate phosphate; b) inulin; c) algammulin which is a combination ofinulin and aluminum hydroxide; d) monophosphoryl lipid A (MPL); e)resiquimod; f) muramyl dipeptide (MDP); g) N-glycolyl dipeptide (GMDP);h) polylC; i) CpG oligonucleotide; j) aluminum hydroxide with MPL; k)any water in oil emulsion; 1) any oil in water emulsion that containsone or more of the following constituents: squalene or its analogues orany pharmaceutically acceptable oil, tween-80, sorbitantrioleate,alpha-tocopherol, cholecalciferol and aqueous buffer, or any of theanalogues and derivatives of the molecules thereof wherein one or two ormore combination of any of the aforementioned adjuvants when formulatedwith Zika, Chikungunya and Japanese encephalitis virus antigens elicitsimmune response against the virus. Preferably, the adjuvant is aluminiumhydroxide at 0.25 mg to 1.0 mg of aluminium content per vaccine dose.

The vaccine composition of the invention optionally comprises2-phenoxyethanol preservative at a concentration of 2.5 to 5 mg/mL.

The vaccine composition when administered in a single dose or in two ormore doses in mammals elicits Th1 and Th2 immune response against any ofthe arbovirus antigens comprising Zika Virus, Chikungunya virus andJapanese Encephalitis virus and is suitable for administration tohumans.

In one embodiment the invention provides a method for preparation of avaccine composition comprising one or more arbovirus antigens selectedfrom Zika virus, Chikungunya virus and Japanese encephalitis virus, themethod comprising one or more steps of inactivation, producingrecombinant protein, expressing structural antigens, purification andconcentration of the virus antigen wherein said purification andconcentration of Zika virus comprises one or more steps selected from:

-   -   a. ultracentrifugation;    -   b. density gradient centrifugation;    -   c. clarification of the viral harvest using membrane filtration;    -   d. purification by column chromatography;    -   e. tangential flow filtration using membranes with cut off from        100 kDa to 300 kDa, wherein tangential filtration is carried out        either before or after virus inactivation.

The column chromatography method comprises gel filtration, mixed moderesin column chromatography, any ion exchange column chromatography,affinity matrix chromatography and hydrophobic interactionchromatography, wherein the column chromatographic method elutesmajority of the virus antigen in the flow through such as Capto Core700, most preferably Capto Core 700 wherein the virus sample is purifiedon Capto Core 700 column and is eluted in the flow through.

The Zika virus is inactivated by one or more inactivating agentsselected from a chemical inactivating agent, a physical inactivatingagent and an irradiating agent.

The preparation method comprises inactivation of Zika virus which may becarried out before or after purification of the virus, wherein the Zikavirus may be inactivated by chemical inactivating agent selected fromformalin (formaldehyde), beta propiolactone (BPL) and hydrogen peroxide.

In one embodiment, the preparation method comprises inactivation of theZika virus bulk which is inactivated by any one of the following methodsselected from:

-   -   a. Formalin treatment at any concentration ranging from 1:500 up        to 1:4000 v/v of formalin:virus, at 8° C. to 37° C., preferably        25±3° C., for at least 1 to 7 days;    -   b. Formalin treatment at any concentration ranging from 1:500 up        to 1:4000 v/v of formalin:virus, at 2° C. to 8° C. for at least        10 to 30 days;    -   c. Beta propiolactone (henceforth BPL) at any concentration        ranging from 1:500 up to 1:4000 v/v of BPL:virus, for at least        24 to 48 hrs, if not more, at temperatures ranging from 8° C. to        30° C., preferably 25±3° C., for 48 hours;    -   d. Beta propiolactone at any concentration ranging from 1:500 up        to 1:4000 (BPL:virus, v/v), at 2° C. to 8° C. for at least 3-7        days;    -   e. a combination of BPL and formalin at any of the        aforementioned conditions, preferably BPL inactivation at 1:3000        (BPL:virus, v/v) for 24 hours followed by formalin inactivation        at 1:3000 (formalin:virus, v/v) for 24 to 48 hours at 15° C. to        30° C., preferably 25±3° C.;    -   f. hydrogen peroxide at any concentration from 0.1 to 3%,        preferably 0.1 to 1% at any temperature from 20-30° C. for 5        minutes to 120 minutes.

In embodiment of preparation method, the virus is inactivated by gammairradiation by exposure from 20 kGy (Kilo Gray) up to 35 kGy, preferably25 kGy to 30 kGy from a ⁶⁰Co source.

In another embodiment of preparation method, the Zika virus isinactivated by UV irradiation by exposure to 254 nm for 30-60 minutes.

In another embodiment of the preparatory method, the Zika virus isinactivated by heat treatment from 50° C. to 65′C for 30 min up to 2hrs, preferably, 65° C. for 1 hr.

In one embodiment of the preparation method, the inactivation is carriedout in the presence of stabilizing agent selected from lactose, sucrose,trehalose, maltose, mannose, iso-maltose, raffinose, stachyose,lactobiose, sorbitol, mannitol, lactobionic acid, dextran, L-glycine,L-histidine, L-glutamic acid, L-aspartic acid and human serum albumin orcombinations thereof. In one preferred embodiment the stabilizing agentis selected from:

-   -   a. 2% sorbitol and 1% L-glycine;    -   b. 1% sorbitol and 0.5% L-glycine;    -   c. 1% mannitol and 0.5% L-glycine;    -   d. 1% mannitol and 0.5% L-glutamic acid; and    -   e. 1% sorbitol and 0.5% L-glycine, 1% human serum albumin.

The inactivation methods described hereinabove are applicable to Zikavirus of any genotype/strain, live attenuated Zika virus, deactivatedvirus, virus like particles, chimeric virus particles that carry anyZika virus antigens particularly the E protein in any heterologous virusbackbone, in vectored vaccines and infectious synthetic virus particlesderived in vitro or in vivo using the sequence of any Zika virus genome.

In one embodiment the invention discloses a method of producing therecombinant protein comprising the steps of:

-   -   a. transfecting recombinant plasmid DNA in insect cells;    -   b. harvesting the cells and isolating the recombinant protein        therefrom;    -   c. purifying the protein by at least one of the methods        comprising of ion exchange chromatography, gel filtration,        affinity chromatography, hydrophobic column chromatography,        mixed mode resin chromatography, diafiltration,        ultracentrifugation, density gradient centrifugation,        fractionation with salt.

In another embodiment, the invention discloses method of expressing thestructural antigens of Zika virus comprising expression system is anyprokaryotic or eukaryotic expression system including baculovirusmediated expression in insect cells.

In another embodiment the invention discloses a method wherein themethod comprises neutralizing antibodies that are largely elicitedagainst the Envelope protein such as in optimally inactivated virus,live attenuated virus, deactivated virus, DNA vaccine, virus likeparticles, chimeric virus particles that display the Zika virus Eprotein in any heterologous virus backbone such as in vectored vaccinesand synthetic virus particles derived from any Zika virus genomic RNAsequence.

The vaccine composition of the invention may be administered in a primeboost strategy, wherein the prime is the candidate inactivated vaccineand the boost is either the same vaccine or any other vaccine such asDNA vaccine, Chimeric Zika virus vaccine, virus like particles,deactivated Zika vaccine, live attenuated virus vaccine, recombinantsubunit vaccine, vectored vaccine or any vaccine derived from syntheticZika virus, wherein the neutralizing antibodies in each of them areelicited against Zika virus Envelope protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Purified Zika virus bulk in 12.5% SDS-PAGE gel detected bysilver staining. The Envelope (E) protein and the Membrane (M) proteinsare the major proteins detected in the purified antigen

FIG. 2A: Inactivation kinetics of Zika virus by formalin atconcentrations ranging from 1:1000 v/v of formalin:virus up to 1:4000v/v of formalin:virus at 25±3° C.

FIG. 2B: inactivation kinetics of Zika virus by beta-propiolactone atconcentrations ranging from 1:1000 up to 1:3500 v/v of BPL:virus at25±3° C.

In both the inactivation procedures, of FIG. 2A and FIG. 2B, 1% sorbitolad 0.5% L-glycine (final concentration) were added as stabilizers, whichhad no effect on the inactivation kinetics. The inactivated samples wereserially amplified three times in vitro in Vero cells, and assayed atthe end of three passages by TCID50.

FIG. 3A: The ˜2.1 kb Zika virus prME gene of SEQ ID NO.1 was amplifiedby gene specific primers for initiating cloning in pFastBac vector forexpression in insect cells.

FIG. 3B: The Sf9 cell lysate was probed by Western for detection ofexpression of the prME protein using Zika rabbit polyclonal antisera bystandard procedures. The envelope protein of 55 kD could be detected asthe major band.

FIG. 4: Estimation of neutralizing antibody titers elicited by Zikavaccine formulations with different adjuvants. Adjuvants are abbreviatedas follows: pIC (polyIC); C—cholecalciferol; MPL (lipid A;monophosphoryl); RP (resiquimod+polyIC); RM (resiquimod+OWEM2); I(inulin); OWEM2 (oil in water emulsion 2); AI (aluminumhydroxide+inulin); MDP (muramyldi peptide); OWEM1 (oil in water emulsion1). No significant antibody titers could be detected in the respectivecontrol groups and hence not depicted in the figure. In all cases, 10 μgof two doses of Zika vaccine antigen was formulated for administrationin Balb/c mice by IM route.

FIG. 5A: Estimation of neutralizing antibody titers by PRNT₅₀ in doseranging studies from 0.125 μg up to 40 μg per dose of the aluminumhydroxide adjuvanted formalin inactivated Zika virus vaccineadministered by IM route in Balb/c mice in two doses.

FIG. 5B: the vaccinated animals were challenged intravenously with 10e5PFU/animal of the Zika virus strain 7 days after the booster dose, andviremia was monitored every 24 hours for 7 days (depicted in the graphfor 6 days). All the animals showed complete protection from viremiawhereas the animals administered the placebo control showed viremia thatpersisted up to 6 days. The infectious virus was estimated in the bloodsamples by TCID₅₀.

FIG. 6: Virus challenge in 4-6 week old Balb/c mice after administrationof 1 μg to 40 μg of BPL inactivated, alum adsorbed Zika virus vaccine.Animals of all the vaccine dose groups and the placebo group werechallenged with 10e5 PFU of Zika virus MR766 strain 7 days afteradministration of the booster dose. Viremia was monitored at 48 and 96hours after virus challenge, and the titers of infectious particles inblood were estimated by TCID₅₀. The candidate Zika vaccine offeredcomplete protection from virus challenge in all the dose groups.

FIG. 7: Passive immunization offered complete protection against viremiaand infectious virus could not be detected by TCID₅₀ in the animals thatreceived Zika rabbit polyclonal antisera intraperitoneally andchallenged 24 later with 10e5 PFU of Zika virus. Infectious virusparticles could not be detected by TCID₅₀ in the blood, when monitoredevery 24 hours for 6 days, whereas the control animals that receivedequal volume of PBS showed persistent viremia up to 6 days whenchallenged with the same dose of the virus.

FIG. 8A: Neutralization of FSS 13025 Zika virus strain by vaccine andplacebo.

FIG. 8B: Neutralization of MR766 Zika virus strain by vaccine andplacebo.

Formalin inactivated, alum adsorbed Zika virus vaccine antisera fromvaccinated mice neutralized the homologous MR766 Zika virus strain (FIG.8B) and cross neutralized the heterologous Asian genotype FSS13025strain (FIG. 8A) with equal efficiency with PRNT₅₀ titers of 18105and 18325 respectively. The values for the placebo (alum only) are alsodepicted in the graph alongside.

FIG. 9A: Antibody titers expressed as log 10 of reciprocal of serumdilutions from the dose ranging studies with single dose of formalininactivated vaccine administered in 4-6 week old Balb/c mice asdescribed in Example 7.

FIG. 9B: Antibody titers expressed as log 10 of reciprocal of serumdilutions from the dose ranging studies with two doses of formalininactivated vaccine administered in 4-6 week old Balb/c mice asdescribed in Example 7.

FIG. 9C: Antibody titers expressed as log 10 of reciprocal of serumdilutions from the dose ranging studies with three doses of formalininactivated vaccine administered in 4-6 week old Balb/c mice asdescribed in Example 7.

FIG. 9D: Antibody titers with a single, two and three doses of 10 μg ofvaccine antigen without alum. All values are expressed as Geometric MeanTiters with 95% CI.

In 9A-9D, individual animal data is plotted. Zika virus antigen wasimmunogenic even without an adjuvant. Data from other dose ranges wereestimated but not included in the graph.

FIG. 10: High affinity antibodies could be elicited by single dose offormalin inactivated alum adsorbed Zika virus vaccine in Balb/c miceeven at low doses of the vaccine antigen up to 1 μg. Antibody aviditywas expressed as avidity index and estimated by methods described inExample 12.

FIG. 11A: Estimation of Th1 cytokine, IFN gamma, in mice vaccinated withZika vaccine formulations with different adjuvants.

FIG. 11B: Estimation of Th1 cytokine, IL-2, in mice vaccinated with Zikavaccine formulations with different adjuvants.

In all cases, in FIG. 11A and FIG. 11B, it was 10 μg of vaccine antigenper dose. Adjuvants are abbreviated as follows: pIC (polyIC);C—cholecalciferol; MPL (lipid A; monophosphoryl); RP(resiquimod+polyIC); RM (resiquimod+OWEM2); I (inulin); OWEM2 (oil inwater emulsion 2); AI (aluminum hydroxide+inulin); MDP (muramyl dipeptide); OWEM1 (oil in water emulsion 1) as described in Example 5. Oilbased adjuvants and polyIC elicited a strong Th1 response compared toother adjuvants tested.

FIG. 12A: Estimation of Th2 cytokine, IL-4, in mice vaccinated with Zikavaccine formulations with different adjuvants.

FIG. 12B: Estimation of Th2 cytokine, IL-10, in mice vaccinated withZika vaccine formulations with different adjuvants.

In all cases, in FIG. 12A and FIG. 12B, it was 10 μg of vaccine antigenper dose. Adjuvants are abbreviated as follows: pIC (polyIC);C—cholecalciferol; MPL (lipid A; monophosphoryl); RP(resiquimod+polyIC); RM (resiquimod+OWEM2); I (inulin); OWEM2 (oil inwater emulsion 2); AI (aluminum hydroxide+inulin); MDP (muramyl dipeptide); OWEM1 (oil in water emulsion 1) as described in Example 5. Oilbased adjuvants and polyIC elicited strong Th2 response in addition toTh1 response.

DETAILED DESCRIPTION OF THE INVENTION

This disclosure concerns formulation of vaccine compositions. Theinvention discloses in particular, preparation and formulation ofvaccine antigens of Zika virus in monovalent compositions and incombination with other arboviruses such as Chikungunya and/or Japaneseencephalitis viruses. In particular, the invention disclosescompositions for prophylaxis and treatment of Zika virus infections.

One aspect of the invention is that the methods of preparation,formulation and use of Zika antigens as vaccine for eliciting immuneresponse is applicable to any genotype, genotypic variants or any strainof Zika virus wherein one genotype of Zika virus cross neutralizes aheterologous strain efficiently. The Zika virus can be selected fromAsian, West African or East African genotype of the virus. Therefore,the methods described in the current invention herein are applicable toZika virus of any genotype/strain, live attenuated Zika virus,deactivated virus, virus like particles, chimeric virus particles thatcarry any Zika virus antigens particularly the E protein and the Mprotein in any heterologous virus backbone, in vectored vaccines andinfectious synthetic virus particles derived in vitro or in vivo usingthe sequence of any Zika virus genome. A chimeric virus has the nucleicacid of a heterologous virus and nucleic acid of Zika virus.

In the context of the immunogenic compositions disclosed herein, inparticular the bulk antigen used for preparation of immunogeniccompositions, the methods of preparation, formulations and use of Zikavaccine antigens are applicable to any of the aforementioned Zika virustypes, that share at least 50% amino acid identity and up to 100% aminoacid identity across any region of the genome. In the context of theimmunogenic compositions disclosed herein, sequence of Zika virus MR766strain of African genotype (SEQ ID NO:5 for genomic nucleotide sequenceand SEQ ID NO:6 for complete ORF) shares more than 96.5% amino acididentity in the structural Envelope protein with the Asian genotypestrain FSS13025 and whose sequence is disclosed in SEQ ID NO:7 and SEQID NO:8 for the nucleotide and protein sequences respectively. Vaccineantisera of the MR766 strain cross neutralized the FSS13025 strain with100% equivalent potency as the homotypic MR766 strain. Also in thecontext of the disclosure herein, Zika virus prME (SEQ ID NO:3) antiseraefficiently cross neutralized the MR766 strain confirming that all Zikaviruses are serotypically similar. In the context of the disclosureherein, the vaccine methods developed using any one of the Zika virusstrains is applicable to homologous and any heterologous Zika virusstrains for use as candidate vaccine.

A cell line that can be propagated in vitro in culture can be used as ahost for Zika virus culture. For propagating Zika virus strains,preferably permissive cells which allow the virus to grow well areselected. For example, diploid cell lines such as MRC-5 and WI-38, andserially passaged cell lines such as Vero, BHK-21, CHO cells etc. can beused. For example, Vero cells (ATCC No. CCL-81), BHK-21 (ATCC No.CCL-10), C6/C3 (ATCC No. CRL-1660) etc. can be used. In a preferredembodiment, one such cell line used in the current invention is Verocells (ATCC No. CCL-81) which has been validated for use as a host cellfor vaccine production. The validated Vero cell lines conforms to theRequirements for Biological Substances No. 50 regarding requirements foruse of cells for the production of biologicals recommended by the WorldHealth Organization (WHO) thereby confirming these cell lines asqualified for producing a vaccine (WHO Technical Report Series, No. 878,pp 19-52, 1998).

In one aspect of the invention, the method of adaptation of Zika virusto Vero cells increases the virus titer. Zika virus passaged repeatedlyin Vero cells increases the virus titer. In the context of virus growthin Vero cells disclosed herein, Zika virus passaged initially in mousebrain or Ae. albopictus C6/36 cells (ATCC No. CRL-160) and then adaptedto Vero cells increases virus titers suitable for vaccine production.

For maintenance in cell culture of the above-mentioned cell lines, Verocells in particular, stationary culture in monolayers, perfusion systemculture, shake flasks, roller tube/bottle culture, suspension culturewith and without microcarriers, cell factories and cell stacks,bioreactors and disposable bioreactors, wave bioreactors and the likecan be adopted. For example, various types of microcarriers arecommercially available. Commercially available animal cell culturedevices can be used to facilitate the growth of cells to high celldensity.

In one aspect of the invention and disclosed herein, the Zika virus ispurified for use as candidate vaccine. Purification is achieved by acombination of both physical and chemical methods either before or afterinactivation of the virus. Physical methods include any of the followingtechniques but not limited to: ultracentrifugation, density gradientcentrifugation, ultrafiltration, diafiltration and concentration usingsemi-permeable membranes with suitable molecular cut-off sizes.Purification through chemical means employs methods such asadsorption/desorption through chemical or physiochemical reactions suchas ion exchange chromatography, affinity chromatography, hydrophobicinteraction chromatography, gel filtration chromatography such as forexample Captocore700™, hydroxyapatite matrix, salting with inorganicsalts, one such example being ammonium sulphate.

In a preferred embodiment, the virus is purified on Capto core 700 (GEHealthcare Life Sciences) column chromatography. Inactivation of thevirus is achieved either before purification or after purification onCapto core 700 column. The virus harvest before Capto core 700 columncan be clarified using membrane filters with different pore sizes,preferably not less than 0.45 μM low protein binding membrane. In apreferred embodiment, the virus harvest can be clarified with a dualmembrane of two different pore sizes, for example 1.2 μM followed by0.45 μM, or 0.8 μM followed by 0.45 μM. The clarified virus harvest issuitable for purification on Capto Core 700 column. The buffers used forpurification on Capto core 700 is of optimal pH and ionic strength tomaximize the binding of the impurities on the column and elute the virusin the flow through. The virus sample is further concentrated bydiafiltration before or after virus inactivation. Diafiltration of thevirus sample after inactivation removes the virus inactivating agentfrom the bulk antigen, and is suitable for formulation.

In one embodiment of the invention, Zika virus in inactivated (killed)for use as a vaccine antigen. Inactivation can be carried out eitherbefore or after purification of the virus. In a preferred embodiment,inactivation of Zika virus is carried out after purification of thevirus.

Zika virus can be inactivated either by heat, gamma irradiation,ultraviolet light or by chemical means. In a preferred embodimentdisclosed herein, Zika virus is chemically inactivated. Chemicalinactivating agents were selected from the following list which includesbut is not limited to: formalin, beta-propiolactone, glutaraldehyde,N-acetylethyleneimine, binary ethyleneimine, tertiary ethyleneimine,ascorbic acid, caprylic acid, psolarens, detergents including non-ionicdetergents etc. wherein the chemical inactivating agent is added to avirus suspension to inactivate the virus.

In a preferred embodiment of the invention, the chemical inactivatingagent selected is formalin and/or beta propiolactone (BPL). Formalin isused at any concentration ranging from 1:1000 to 1:4000 v/v offormalin:virus. Beta propiolactone is used at any concentration rangingfrom 1:1000 to 1:4000 v/v of BPL:virus. The temperature and duration ofinactivation is optimized to complete virus inactivation with minimaladverse effect on immunogenicity. This can be achieved with shorterduration of exposure with minimum quantity of the inactivating agent. Inthe context of virus inactivation, the disclosure herein describes theconcentration, temperature and time of exposure of Zika virus toformalin and BPL. In the preferred embodiment of the invention, theinactivation temperature is 25±3° C., most preferably 22° C. for 7 days.At lower temperatures of 2° C. to 8° C., the duration of formalinexposure is longer than 7 days to achieve complete virus inactivation atthe aforementioned concentration ranges. Duration of virus exposure toformalin can be reduced to below 48 hours by increasing the temperatureof exposure up to 37° C. Hence, effective formalin inactivation of Zikavirus can be achieved at any concentration range of formalin from 1:1000v/v formalin:virus up to 1:4000 v/v formalin:virus by choosing anytemperature range from 2° C. to 37° C. and varying the exposure timefrom 24 hours to more than 10 days at any of the aforementionedconcentrations, time and temperature of exposure.

In one embodiment of the disclosure, BPL is used as virus inactivatingagent for Zika virus. In a preferred embodiment of the invention, BPL isused at concentrations ranging from 1:1000 v/v BPL:virus up to 1:4000v/v BPL:virus. At lower temperatures of 2 to 8° C., the duration of BPLexposure is preferred for 3 to 7 days to achieve complete virusinactivation at the aforementioned concentration ranges. Duration ofvirus exposure to BPL can be reduced to 48 hours or below by increasingthe temperature of exposure up to 25±3° C. or even up to 37° C. Hence,effective BPL inactivation of Zika virus can be achieved by choosing anyconcentration range of BPL from 1:1000 v/v BPL:virus to 1:4000 v/vBPL:virus by choosing any temperature range from 2 to 37° C. and varyingthe exposure time from 24 hours to more than 10 days at any of theaforementioned concentrations, time and temperature of exposure.

One of embodiments of the current invention disclosed herein is the useof a combination of BPL and formalin at any of the aforementionedconditions, preferably BPL inactivation at 1:3000 v/v of BPL:virus for24 hours followed by formalin inactivation at 1:3000 v/v formalin:virusfor 24 to 48 hours at 15° C. to 30° C., preferably 25±3° C. The use ofBPL and formalin combination for Zika virus inactivation is that themechanism of inactivation being different for formalin and BPL, theircombined use reduces their overall concentration and exposure to boththe inactivating agents, and also the use of low concentrations offormalin promotes stability of the virus bulk by promoting cross linkingof virus epitopes. In another embodiment of the invention, hydrogenperoxide is used for inactivating the Zika virus at concentrationsranging from 0.1 to 3%, preferably 0.1 to 1% at any temperature from 20°C. to 30° C. for 5 to 120 minutes, if not more.

An embodiment of the current invention discloses the use of the prME(pre-membrane and the Envelope protein) of Zika virus as the candidatevaccine antigen to elicit immune response against the Zika virus. Thedisclosure is applicable to any method of vaccine design wherein theprME or the E protein is expressed in such a manner that theneutralizing Zika antibodies are directed against the said antigens. Ina preferred embodiment of the invention, the prME protein is expressedas recombinant virus like particles (VLP) in baculovirus mediatedexpression in insect cells. Anyone skilled in the art will deriveadditional embodiments using the above disclosure to design a vaccinecandidate using the prME protein as the target Zika antigen such as aDNA vaccine, Virus like particles comprising prME proteins, subunitvaccine comprising the Envelope (E) antigen, live vectored vaccines,chimeric vaccines using the Zika prME on a heterologous nucleic acidbackbone wherein in all the above, the anti-Zika antibodies are largelydirectly against the E protein.

In the current invention disclosed herein, are immunogenic compositionscomprising purified recombinant Zika virus antigens comprising theenvelope (E) protein, membrane (M) protein and optionally thenon-structural 1 (NS1) protein as vaccine antigens for eliciting immuneresponse for prophylaxis of Zika virus infections. In a preferredembodiment, the use of the Zika virus having of the prME gene ofsequences SEQ ID NO:1 and SEQ ID NO:2 encoding the structural protein ofSEQ ID NO:3 and SEQ ID NO:4 respectively, wherein the expressed andpurified prME protein can be used as vaccine antigen for prophylaxis ofZika virus infections.

In a preferred embodiment, Zika virus prME gene is used to generate arecombinant gene construct that can be used to express the prME proteinin prokaryotic or eukaryotic expression systems as virus like particles(VLPs), preferably baculovirus mediated expression in insect cells. Themethods disclosed herein are applicable to any Zika virus strain thatshare at least 70% amino acid identity to the aforementioned SEQ ID NO:3and SEQ ID NO:4

An embodiment of the current disclosure is the choice ofpharmaceutically acceptable buffer throughout the bioprocess wherein thebuffering agent is selected from a list consisting of any one or more ofthe following, but not limited to: phosphate buffer; citrate buffer;phosphate citrate buffer; borate buffer; tris(hydroxymethyl)aminomethane(Tris) containing buffer; succinate buffer; buffers containing glycineor histidine as one of the buffering agents. In the most preferredembodiment, phosphate buffer is used, wherein phosphate buffer is sodiumphosphate buffer at concentration of 5 mM up to 200 mM of phosphateions, preferably 10 mM to 100 mM phosphate buffer, most preferably 10 mMto 50 mM phosphate buffer of any pH above 6.50 to pH 9, preferably pH6.8 to pH 7.8 is used for the upstream and downstream processes. In apreferred embodiment, 10 mM sodium phosphate buffer of pH 7.4±0.2 isused in the preparation of the purified inactivated vaccine bulk of Zikavirus antigen, and optionally containing sodium chloride at aconcentration from 50 to 200 mM. In another preferred embodiment,sorbitol and L-glycine are optionally added to a final concentration of1% and 0.5% respectively.

An embodiment of the current invention also discloses the choice ofadjuvants that is compatible for formulation with Zika virus antigen.

The antigenic compositions of Zika virus as monovalent vaccine, and withChikungunya virus and Japanese encephalitis viruses in combinationvaccine were formulated in pharmaceutically acceptable carrier forimmunization. The use of adjuvant(s) can reduce the amount of antigenrequired in the formulation Furthermore, for adjuvanted vaccineformulations, suitable adjuvant(s) were selected from the followinglist, which includes but is not limited to: alum such as aluminumhydroxide, aluminum phosphate, or amorphous aluminum sulphate phosphate;calcium phosphate; inulin of any polymorphic form, preferably gammainulin; adjuvants containing inulin in combination with other organicand inorganic compounds such as aluminum hydroxide, aluminum phosphate,aluminum sulphate phosphate and calcium phosphate; liposomes, chitosanand complex carbohydrates such as dextran, dextrins, starch, mannans andglucomannans, galactomannans, beta-glucans, heparin, cellulose,hemicellulose, pectins and pectinates, lectins and any othercarbohydrates either synthetic or derived from any source, anybiodegradable and biocompatible polymers, such as poly lactide andpolylactide co-glycolides, (PLG or PLGA); any emulsions including butnot limited to oil in water emulsions one such example being squalene orsqualene analogues containing oil in water adjuvants, oil in wateremulsions containing vegetable oils; any water in oil emulsion;liposomes prepared with cholecalciferol as one of the ingredients alongwith other lipid soluble compounds; liposomes of other compositions;RIBI adjuvant systems, saponins including but not limited to QS-21,QuilA, tomatine, ISCOMs, ISCOMATRIX etc, lipopeptides, glycopeptides andtheir analogues, resiquimoid, lipopolysaccharides, lipid A, muramyldipeptides or their analogues and any peptide based adjuvants,oligonucleotides, any TLR ligands and their analogues as adjuvants, anycytokine, vitamins and non-toxic bacterial toxins, indeed any analoguesof all the aforementioned adjuvants and combination of two or more ofthe aforementioned adjuvants or their analogues that are compatible invaccine formulation(s). and tested for enhanced immunogenicity. Inaddition to the above, any other organic and inorganic substances thathave good immunopotentiating activity are suitable to be used asadjuvant either singly or in adjuvant combinations to enhance theimmunogenicity of the arboviral antigens. The use of adjuvant in thevaccine formulations can reduce the amount of antigen required.

In a preferred embodiment of the invention, aluminum hydroxide was usedfor dose ranging studies of both formalin and BPL inactivated Zikaantigens as well in vaccine combinations of Zika, CHIKV and JEV vaccinesdue its safety profile for use in target population. Oil based emulsionsand polylC gave good immunopotentiating effect to Zika antigen when usedas adjuvants. In one embodiment of invention, polylC and other adjuvantsthat offer both systemic mucosal immunity is particularly advantageousfor protection against disease caused by Zika virus infections. PolyICand the oil based emulsions and the adjuvant combinations disclosed inthe invention elicited both Th1 and Th2 responses estimated by themeasurement of the Th1 and Th2 cytokines after vaccination. Several ofthe above mentioned adjuvants also elicit strong mucosal immunity inaddition to systemic immunity, one such example being polylC. Zikavaccine antigens either the inactivated or purified recombinant prMEproteins are formulated with any of the adjuvants that elicit bothsystemic and mucosal immunity.

In one embodiment of the current invention, a vaccine preservative isused in the vaccine formulations. The preferred embodiment is 2-phenoxyethanol at a concentration of 2.5 to 5 mg per dose 2.5 to 5 mg per mL

In one aspect of the current invention disclosed herein are the use ofstabilizing agents selected from one or more of the following, but notlimited to: lactose, sucrose, trehalose, maltose, mannose, iso-maltose,raffinose, stachyose, lactobiose, sorbitol, mannitol, lactobionic acid,dextran, L-glycine, L-histidine, L-glutamic acid, L-aspartic acid, humanserum albumin and combinations thereof, at any suitable concentrationthat are used to confer stability during the inactivation of Zika virusby any of the aforementioned methods. In a preferred embodiment, thestabilizing agents are selected from any of the following combinationsbut not limited to: 2% sorbitol and 1% L-glycine; 1% sorbitol and 0.5%L-glycine; 1% mannitol and 0.5% L-glycine; 1% mannitol and 0.5%L-glutamic acid; 1% sorbitol, 0.5% L-glycine, 1% human serum albumin. Ina preferred embodiment, the combination of 1% sorbitol and 0.5%L-glycine and 1% mannitol and 0.5% L-glycine are preferred combinations,most preferably, 1% sorbitol and 0.5% L-glycine. One skilled in the artwill recognize further embodiments based on the above disclosures.

Lyophilized formulations are one of the methods for preparation ofvaccine product. Lyophilized preparations of Zika virus vaccinetypically contain purified inactivated Zika virus, a sugar polyol,preferably sorbitol and mannitol, most preferably sorbitol incombination with a glass forming sugar, which is preferably adisaccharide or an oligosaccharide. The preferred disaccharide isselected from the following list but is not limited to: sucrose,trehalose, maltose, mannose, lactose, raffinose, isomaltose, stachyoseetc. the preferred embodiment of the disclosure is a combination of 1%sorbitol with 5% sucrose, 1% mannitol with 5% sucrose, and 3% sucroseand 2% trehalose, 1% mannitol with 1% L-glycine and or 2% trehalose. Anyone of the ordinary skill in the art will devise further embodiments andbased on the disclosures above.

The lyophilized formulations can be re-suspended in water for injectionor an aqueous buffer that is pharmaceutically acceptable foradministration. e.g. as an injectable liquid to a human subject. Thelyophilized formulation can also be used as an inhalable powder whichwill be suitable for inducing mucosal immunity. Additionally, thelyophilized formulation of Zika virus can comprise an adjuvant thatconfers mucosal immunity preferably from a list of those adjuvantstested in the current invention for Zika virus such as polylC forexample.

In the current invention, the disclosure provided herein on the optimaluse of Zika virus antigen to elicit robust immune response, the vaccineantigen can be used at 0.10 μg up to 100 μg per dose, wherein thepreferred embodiment is any concentration from 0.125 μg up to 40 μg perdose such that the administered vaccine doses elicit antibody titersmeasurable by assays such as ELISA and PRNT₅₀. The vaccine can beadministered with and without an adjuvant as both the inactivatedvaccine and the adjuvanted formulations elicit good immune response.

In yet another disclosure of the invention, the inactivated Zika vaccinecandidate inactivated by any of the disclosed methods can beadministered as a single dose or in two or more doses to elicit immuneresponse. The methods disclosed in the invention provide the kinetics ofimmune response after each dose of the vaccine, at dose ranges from0.125 μg up to 40 μg per dose that offers the flexibility of the choiceof the vaccine dose range concentrations and number of doses to suit thetarget population for vaccination.

The route of vaccine administration can be by any route selected from,but not limited to intramuscular, intradermal, subcutaneous,intravenous, oral, intranasal and transcutaneous routes. In a preferredembodiment of the invention, the preferred route of vaccineadministration is intramuscular (IM) route.

The vaccine formulations can be presented in glass vials and injected byneedle and syringes, presented in pre-filled syringes in a ready to usepresentation or administered by electroporation, microneedle patches,needle free patch, by inhalation or by nasal sprays.

The current invention discloses methods for preparation and use offormulations comprising one or more arbovirus antigens selected from alist that includes Zika virus, Chikungunya virus (CHIKV), and Japaneseencephalitis virus (JEV). When used in vaccine combination, the vaccinecan elicit immune response against each of the viruses present in acombination vaccine. In a preferred embodiment of the inventioncomprising a vaccine composition wherein Zika virus antigens andJapanese encephalitis virus antigens are present in a combinationvaccine at concentrations ranging from 5 μg to 50 μg of each antigen ina pharmaceutically acceptable formulation without an adjuvant, orpreferably with an adjuvant selected from the list of adjuvantsdisclosed in the current invention, preferably aluminum hydroxide with0.25 mg to 1.5 mg of aluminum content per vaccine dose is disclosed. Inyet another preferred embodiment of the invention a vaccine compositioncomprising Chikungunya and Zika virus antigens in a formulationcomprising 5 μg to 50 μg of each antigen in a pharmaceuticallyacceptable formulation without an adjuvant, or preferably with anadjuvant selected from the list of adjuvants disclosed in the currentinvention, preferably aluminum hydroxide with 0.25 mg to 1.5 mg ofaluminum content per vaccine dose is disclosed.

In yet another preferred embodiment of the invention, a vaccinecomposition comprising Chikungunya, Zika and JEV virus antigens in aformulation comprising 5 μg to 50 μg of each antigen in apharmaceutically acceptable formulation without an adjuvant, orpreferably with an adjuvant selected from the list of adjuvantsdisclosed in the current invention, preferably aluminum hydroxide with0.25 mg to 1.5 mg of aluminum content per vaccine dose is disclosed. Theuse of vaccine combination confers a distinct economical advantage formanufacture and distribution of vaccines, provided that immune responseis elicited against each of the antigen in the formulation and noantigenic interference is observed to either of the antigen by thepresence of an additional antigen. The vaccine antigens can either beadministered from a single formulation or administered separately at thesame time or in suitable time intervals so as to elicit an immuneresponse to the cognate antigen. The recombinant Zika prME protein canbe used in vaccine combination with inactivated JE and CHIKV vaccines inlieu of inactivated Zika virus vaccine.

Recombinant CHIKV VLP obtained by expressing the structural polyproteinof CHIKV comprising largely of the capsid, E2 and E1 and 6K proteins orE2, E1 and 6K polypeptides or E2 and E1 can be used in combination withinactivated Zika and JE vaccines as a combination.

The current invention also discloses the use of Zika virus antibodiesfor detection of Zika virus by ELISA or in any immunodiagnostic methodswhere the antibodies find an application for detection or diagnosis ofZika virus infections.

The current invention also discloses herein the use of Zika virusantibodies for prevention and treatment of Zika virus disease.

Abbreviations used in the invention: IM—intramuscular; mcg-microgram;TCID50-50% Tissue Culture Infectious Dose; PFU—Plaque forming unit

EXAMPLES Example 1: Zika Virus Culture in Vero Cells

Vero cell line (ATCC No. CCL-81) was used as the cell substrate forculture of Zika virus.

Extensively characterized Vero cells obtained from BioReliance, USA wasused in pilot scale production. Vero cells were grown in DMEM(Dulbecco's Modified Eagle Medium; Sigma-Aldrich Catalog #D5523 and usedas per the manufacturer's instructions) or EMEM (Eagles MinimalEssential Medium) containing 5% fetal bovine serum (FBS) or New BornCalf Serum (NBCS) and incubated at 35° C. to 37° C. until reaching80-100% confluence of the monolayer. Post-infection, the same mediumcontaining 1% serum was used, or alternatively the virus was cultured inVero cells adapted to serum free medium. Zika virus also could be grownin MRC-5 cell monolayer which were prepared in growth medium consistingof EMEM buffered to neutral pH with Hepes buffer with 5% serum andstatically incubated at 35° C. to 37° C. for 6 to 8 days. Zika virus wascultured routinely in Vero cells. Zika virus MR766 strain (ATCC VR-84)procured from LGC Promochem, Bangalore was adapted to Vero cells bydirect inoculation in Vero cells. Alternatively, the virus was adaptedin C6/36 Ae. albopictus cells twice by serial passages, and the Zikavirus in culture supernatant from these cells was used to infect Verocells. Serial passage of Zika virus in C6/36 cells cultured at 25° C. to28° C. increased the virus titer higher than 10e8.0 TCID₅₀/mL or 10e8.0PFU/mL. This also obviated the need for subsequent repeat passages inVero cells to obtain high titers. Virus adaptation by this method isuseful to achieve high titers and subsequent higher yield in production.After culture in C6/36 cells, the virus was serially plaque purifiedtwice from Vero cells, and the virus from a single well isolated plaquewas amplified and extensively characterized to be free of adventitiousagents (all known RNA and DNA viruses, bacteria, fungi, mycoplasma etc)using the NGS (Next Generation Sequencing) platform. The virus genomicRNA was sequenced by NGS platform, and complete nucleotide sequence ofMR766 strain is provided in SEQ ID NO:5 and the corresponding deducedamino acid sequence is provided in SEQ ID NO:6. Sequencing showed theintact glycosylation site in the Envelope protein, which otherwise islost if the cells are extensively passaged in mammalian cells. Zikavirus produces cytopathic effect (CPE) in Vero cells, and at the optimalMultiplicity of Infection (MoI) and harvest conditions, virus titersabove 10e8.5 TCID50/mL or 10e8.5 PFU/mL could be attained.

Example 2: Zika Virus Purification

For Zika virus culture at pilot scale, the virus culture wassystematically scaled up from T-175 flasks to CS1 (cell stack 1), CS10(cell stack 10) and CS40 (cell stack 40). Multiples of CS40simultaneously infected with the virus at standardized MoI was used toscale up production. Use of multiples of CS40 enables quick and linearscale up to the desired volumes of production. The harvest volume fromeach CS40 was approximately 8-10 L. The virus was harvested at days 4-6or whenever more than 90% CPE was achieved. Alternatively, disposablebioreactors under well standardized conditions of temperature 35° C. to37° C., pH not less than 7.0, and optimally at pH 7.4, dissolved oxygenat 45 to 75 ppm, preferably 60 rpm and an agitation of 240 to 280 rpmand optimally controlled in-flow and out-flow rate optimized accordingto the scale of the culture volume from 1 L to 100 L was used toincrease the cell density and virus harvest. The viral harvest wasclarified either by microfiltration or using dual filters with cut offof 1.2 μM and 0.45 μM. The clarified viral harvest was then passedthrough Capto Core700 column (GE healthcare Life Sciences) in phosphatebuffered saline, pH 7.4. The Zika virus containing fractions in the flowthrough was optionally concentrated by diafiltration using either 100kDa or 300 kDa cut off membranes. The concentrated virus fraction wasused for virus inactivation. In an alternate method, the clarified viralharvest was inactivated with either BPL or formalin according themethods described in the succeeding sections and then loaded on thecolumn. The purity of the virus was checked on 12.5% SDS-PAGE. There wasno significant difference in the yield or in purity in inactivating thevirus before and after purification. The virus could also be purifiedusing cellufine sulphate, DEAE-Sephadex CM-sephadex with salt gradientand by gel filtration on Sepharose CL-4B, ceramic hydroxyapatite columnwith gradient of 0.2M to 0.8M phosphate and in all cases followed bydiafiltration using 100 or 300 kDa cut off membranes. The purity of thevirus preparation was checked by silver staining of the virus sample in12.5% of SDS-PAGE gel (See FIG. 1). Zika virus by the aforementionedmethods could be purified to high purity suitable to be used as vaccinebulk antigen. The virus could also be purified by ultracentrifugation ona 20-60% sucrose gradient using P28S rotor in Hitachi HIMACultracentrifuge after centrifugation at 100,000×g for 6 to 8 hours.

Example 3: Zika Virus Inactivation

Zika virus sample was inactivated (killed) by various methods for use asvaccine antigens. Formalin inactivation was tested at variousconcentrations ranging from 1:1000 (formalin:virus, v/v) to 1:4000(formalin:virus, v/v) at temperature 25±5° C., more specifically at 22°C. and the kinetics of virus inactivation was monitored every 24 hoursfor up to 10 days, and routinely the virus inactivation was carried outat 25±3° C., preferably at 22° C. for 7 days. The virus inactivation waseffective at all concentrations from 1:1000 v/v formalin:virus, up to1:3500 v/v formalin:virus, at the aforementioned temperatures and timeintervals. A ratio of 1:4000 v/v of formalin:virus was effective invirus inactivation at higher temperatures up to 30 to 37° C. for 3 to 7days. Formalin inactivation was effective at all the aforementionedratios of formalin to virus at temperatures ranging from 2-8° C. whenincubated for time intervals longer than 10 days. Hence formalininactivation offers flexibility of virus inactivation at any temperaturefrom 2° C. to 37° C. at time intervals ranging from 24 hours to morethan 10 days depending upon the conditions used for inactivation. Zikavirus inactivation with Beta propiolactone (BPL) was tested undervarious conditions. Zika virus was completely inactivated at BPLconcentrations ranging from 1:1000 (BPL:virus, v/v) up to 1:3500(BPL:virus, v/v) at temperatures from 25±5° C. for 24 to 48 hours. Athigher concentration of BPL or at higher temperatures up to 37° C.,complete inactivation was achieved in 24 hours or less, and can be usedas a method for quick inactivation of the virus. Zika virus could alsobe inactivated at the aforementioned concentrations of BPL whenincubated at 2 to 8° C. for 3 to 7 days. A combination of BPLinactivation at 1:3500 (BPL:virus, v/v) at 22-25° C. for 48 hours,followed by treatment with low concentrations of formalin from 1:3000 to1:4000 v/v of formalin:virus for 24 hours was effective in bothinactivating and stabilizing the virus. Any concentration of BPL andformalin could be used for both inactivation and stabilizing the virus,as long as inactivation is complete without deleterious effect onimmunogenicity. Inactivation was tested from 0.005% up to 3% finalconcentration of Hydrogen peroxide at 20° C. to 25° C. for a period 2hours. There was no deleterious effect on the immunogenicity of thevirus at lower concentrations of hydrogen peroxide with very briefexposure times within minutes but was deleterious at prolongedconcentrations at higher dose ranges tested. The inactivated virussamples after exposure to different time and dose concentrations weretitered for infectious virus particles if any, by TCID50/mL from 5minutes up to 6 hours at intervals of 5, 10, 20, 30 and 60 minutes andat 2, 4 and 6 hours. At higher concentrations, the virus was inactivatedwithin seconds. At each time point, the reaction was stopped by additionof 10 U/mL of catalase that rapidly hydrolyses hydrogen peroxide. Theoptimum concentration for inactivation was 0.01% final for duration of60 minutes or less as determined by titration for infectious particlesby TCID50/mL and subsequent immunogenicity. Zika virus inactivation withhydrogen peroxide offers the flexibility of duration of exposure atdifferent concentrations for different time points according to theconcentration of virus particles in the sample.

The purified Zika virus sample was heat inactivated at temperatures 50°C. to 65° C. for up to 60 min. UV inactivation of the virus was carriedout UV exposure at 254 nm for up to 120 minutes. Zika virus wasinactivated by gamma irradiation by exposure from 20 kGy (Kilo Gray) upto 35 kGy from a ⁶⁰Co source at the Gamma Agro Medical ProcessingFacility at Hyderabad. All the above inactivation methods were carriedout in the presence and absence of virus stabilizing agents such asvarious concentrations of sugars such as sucrose, lactose, trehalose,maltose, mannose among others. The sugar alcohols used for conferringstabilizing effect were sorbitol and mannitol. The amino acids testedwere selected from L-Histidine, L-Glutamic acid, L-Glycine andL-Aspartic acid and L-Glutamine and also human serum albumin and acombination of one or more of the aforementioned stabilizing agents. Themost effective stabilizing agents were sorbitol at 0.5% to 2%,preferably 1.0% in combination with L-Glycine from 0.5% to 2%,preferably at 0.5%. Mannitol and L-glycine in combination was effectivein stabilizing the virus sample during inactivation rather than Mannitoland L-glycine alone.

The Zika virus samples inactivated by all the aforementioned methods foruse vaccine antigens were tested for completeness of inactivation byserially passaging the inactivated samples three times serially in Verocells and testing for infectious virus at the end of inactivation periodby TCID₅₀. In addition to that, the inactivated virus sample after threeserial passages in vitro was injected intracranially in 2-day old miceand observed for mortality or growth abnormalities for 21 days andconsidered completely inactivated when it showed no adverse effects invitro and in vivo testing. No infectivity was observed with the formalinand beta-propiolactone inactivated virions at the aforementioned rangeof concentrations and for the various time periods tested. Theinactivation kinetics of Zika virus by formalin and BPL as arepresentative example of one of the methods disclosed above is providedin FIG. 2 (FIG. 2A and FIG. 2B)

Example 4: Recombinant Cloning and Expression of Zika Virus pRME Protein

Synthetic gene of the nucleotide sequence SEQ ID NO:1 encoding the OpenReading Frame (ORF) of the prME protein of SEQ ID NO:3 of Zika virus wassynthesized at GenScript, NJ, USA. The gene was PCR amplified with theprimers listed below to obtain a ˜2.1 kb fragment of SEQ ID NO:1encoding the prME protein of SEQ ID NO:3. See FIG. 3A.

FVFP: 5′AACTGCTCGAGGAATTCGGATCCAAC 3′FVRP: 5′ AATGGGCATGCCTGCAGGCGGCCGCTC 3′

The PCR amplified fragments was digested with EcoR1 and Not1 restrictionenzymes and cloned into the EcoR1 and Not1 sites of the pFastBac plasmidvector (Life Technologies, Carlsbad, Calif., USA) under the control ofthe polyhedron promoter by the methods described in the User's manual ofBac to Bac Baculovirus expression system (“An efficient site-specifictransposition system to generate baculovirus for high-level expressionof recombinant proteins, Life Technologies, USA). In brief, the methodutilizes a site specific transposition of the expression cassette suchas the recombinant pFastBac vector with the cloned inserts as describedabove into a baculovirus shuttle vector (bacmid) propagated in E. coli.Recombinant pFastBac vector containing one of the inserts SEQ ID NO:1 orSEQ ID NO:2 cloned under the control of the polyhedron promoter istransformed into competent cells of E. coli Max Efficiency DH10Bac™,that contains a baculovirus shuttle vector (bMON14272) and a helperplasmid (pMON7124) that facilitates transposition to allow efficientre-generation of the recombinant bacmid The recombinant bacmids wereselected on ampicillin, gentamicin and kanamycin containing plates byblue/white selection using bluo-gal or X-gal, and IPTG. The recombinantbacmids after confirmation by PCR for the presence of the gene insertswas isolated by standard protocols described in the aforementioned Usermanual. About 1 μg of the bacmid DNA was used for transfection withLipofectamine in Spodoptera frugiperda Sf9 insect cells (LifeTechnologies, Carlsbad, USA) grown in serum free insect cell medium. Themethods used for transfection, isolation and titration of P1 viralstocks are exactly as described in the User's manual of Bac-to-BacBaculovirus Expression system as given above. The P1 stocks wereserially amplified twice to obtain high titer P3 stocks for expressionof the recombinant prME proteins in Sf9 cells. High titer baculovirusstocks for expression of the prME protein of SEQ ID NO:3 was expressedin 25 mL suspension culture of Sf9 cells and was further scaled upsystematically up to 125 mL per 500 mL flask. Baculovirus infected cellsfrom multiple flasks were harvested at 72 hours post-infection, pooled,washed once with 1×PBS, pH 7.6 and lysed in cell lysis buffer containing10 mM phosphate, pH 7.6 with 50 mM NaCl, 1 mM PMSF and 5 mM EDTA. Thecell lysate was centrifuged at 20,000 rpm for 30 minutes to remove thecell debris and the supernatant was concentrated using proteinconcentrators with 10 kDa cut off membrane. The concentrated sample waslayered on pre-equilibrated 20% to 60% sucrose gradient and centrifugedat 100,000×g for 6-8 hours using P28S rotor in HitachiHIMACultracentrifuge Fractions containing the recombinant Membrane andEnvelope protein was isolated and confirmed by Western blot (FIG. 3B)using the rabbit MR766 polyclonal antisera. The purified recombinantprotein is of the sequence of the contemporary Asian genotype of Zikavirus expressed using the gene sequence SEQ ID NO:1, encoded the proteinof SEQ ID NO:3. The recombinant ME protein cross reacted with MR766antibodies in Western blot and in ELISA and was formulated as vaccineantigen for testing in Balb/c mice as described in sections below.

Example 5: Vaccine Formulations

Zika virus vaccine antigen of any of the aforementioned methods in thepreceding Examples was tested for immunogenicity in laboratory animalswith and without adjuvants. High binding (>95%) was observed to aluminumhydroxide (Alhydrogel® 2%, Brenntag) as the adjuvant, used at the doserange of 0.1 mg to 1.5 mg of aluminum (provided as aluminum hydroxide)per dose even when tested at the high antigen dose of 40 mcg. Bindingwas complete at all the concentrations of Zika virus antigens as well asvaccine combinations with CHIKV and JE antigens discussed in thesucceeding sections that were used for testing in mice. Binding toaluminum hydroxide was carried out for three hours at ambienttemperature. An aliquot of the formulation was centrifuged at 5000×g for5 min and the supernatant was tested for completeness of binding byantigen ELISA. The binding of the antigen was complete as it could notbe detected in the supernatant by ELISA. The buffer for the adjuvantedformulations was 10 mM phosphate buffer, containing 154 mM NaCl, pH7.40±0.2 and optionally containing 1% sorbitol and 0.5% L-Glycine. Otherbuffers used for specific formulations are mentioned below. Theadjuvants listed below were tested for comparative immunogenicity and inall cases concentrations are provided per dose of the vaccine.Inactivated Zika virus antigen was tested at 10 μg per dose:

-   -   a) Inulin (Orafti-HPX, Beneo) was tested at 0.5 mg per dose;        gamma inulin was prepared by the methods described in (Cooper        and Steele, 1988)    -   b) A combination of aluminum hydroxide and inulin. A combination        of inulin and aluminum hydroxide, algammulin was prepared at a        ratio of 10:1 (10 mg/mL inulin: 1 mg/mL aluminum as aluminum        hydroxide) was tested at 0.5 mg per dose    -   c) Muramyl di peptide (L18-MDP) (tlrl-Imdp, Invivogen) at 10 μg        per dose    -   d) MPL (lipid A, monophosphoryl from Salmonella enterica,        L-6895-1 MG, Sigma Aldrich) at 25 μg per dose    -   e) Combination of 0.25 mg aluminum (as aluminum hydroxide) and        25 μg of MPL per dose    -   f) Oil in water emulsion (OWEM1) containing 9.75 mg of squalene        (53626-100ML, Sigma Aldrich), 11.86 mg of alpha-tocopherol        (T3251-5G, Sigma Aldrich), 4.58 mg of Tween-80 (61771205001730,        Merck) in 10 mM phosphate buffer, pH 7.4±0.2.    -   g) Oil in water emulsion 3 (OWEM2) containing 9.75 mg squalene,        1.175 mg of tween-80, 1.175 mg Span-85 (S7135-250ML, Sigma        Aldrich) in 10 mM citrate buffer, pH 7.0    -   h) Poly IC (polyinosinic polycytidylic acid, potassium salt,        Cat. NO. P9582-5MG, Sigma Aldrich) at 25 μg per dose    -   i) Cholecalciferol (Arachitol, Abbot) at 0.75 mg per dose    -   j) Resiquimod (SML0196-10MG, Sigma Aldrich)+Poly IC, 25 μg each    -   k) Resiquimod (25+Oil in water emulsion 2 containing 9.75 mg        squalene, 1.175 mg of tween-80, 1.175 mg Span-85 (S7135-250ML,        Sigma Aldrich) in 10 mM citrate buffer, pH 7.0    -   l) Aluminum 0.25 mg and 0.5 mg per dose provided as aluminum        hydroxide

All the above formulations elicited high level of neutralizingantibodies and the results are depicted in FIG. 4. The individualcomponents of the aforementioned adjuvants and any of their analogues,derivatives, side chain substitutions and any modifications of any ofthe above components at varying concentrations can be used as non-toxicvaccine adjuvant components as long as they have immunopotentiatingeffect. Formalin inactivated and recombinant Zika vaccine antigens asdescribed in the aforementioned sections each at a concentration of 10μg per dose was lyophilized in combination with either of the followingexcipients: 1% mannitol and 0.5% Glycine, 5% sucrose and 1% trehalose,5% sucrose and 1% maltose and 2% mannitol and 0.5% Glycine. The drylyophilized formulation could be easily reconstituted in aqueoussolution with water, normal saline and 10 mM phosphate buffered saline,pH 7.4±0.2. The stability of the formulation was tested at 37° C. fortwo weeks. No change in the cake characteristics was observed indicatingthe stability of the formulations. The moisture content was below 1%.

Example 6: Effect of Stabilizing Agents

The stability of the formalin inactivated vaccine bulk for use asnon-adjuvanted vaccine antigen was tested for stability with thefollowing concentration of stabilizing agents: a) 2% sorbitol and 1%L-glycine; b) 1% sorbitol and 0.5% L-glycine c) 1% mannitol and 0.5%L-glycine; d) 1% mannitol and 0.5% L-glutamic acid e) 1% sorbitol and0.5% L-glycine, 1% human serum albumin. Stability testing was done at37° C. for 2 weeks and the antigen concentration was tested by ELISAbefore and after exposure at 37° C. 1 μg and 10 μg of the non-adjuvantedformulation with 1% sorbitol and 0.5% L-Glycine was tested forimmunogenicity in Balb/c mice as discussed in the succeeding sections.

Example 7: Potency Testing of Vaccine Formulations in Animal Models

Zika vaccine antigen inactivated by the aforementioned methods wastested in Balb/c mice in dose ranges from 0.125 μg up to 40 μg ofantigen per dose with 0.25 mg aluminum per dose (as aluminum hydroxide)in a volume of 100 μL (injected in two sites at 50 μL per site) byintramuscular route on days 0, 14, 28. Initial testing on the effect ofaluminum (provided as aluminum hydroxide showed that alum adsorbedvaccine gave higher titer of neutralizing antibodies than non-adjuvantedvaccine. About 1 and 10 μg of inactivated vaccine antigen without alumcontained 1% sorbitol and 0.5% L-glycine as the excipients to conferstability to the vaccine antigens. Blood was drawn from retro-orbitalsinus on days 13, 21 and 35 for estimation of neutralizing antibodytiters by PRNT₅₀, total Ab titer by ELISA, Ab avidity and cytokineprofiles. Blood withdrawal and testing after each dose gave data on thepotency and safety of single, two doses and three doses of the vaccinepreparations. The animals were each challenged on day 36 with 10e5 PFUof Zika virus by intravenous route. The blood samples were monitored forup to 7 days at 24 hour intervals for formalin groups and at two pointsat 48 hours and 96 hours for BPL inactivation groups for protectionagainst viremia by TCID₅₀ (50% Tissue Culture Infectious Dose) and thevirus titers if any, were expressed as TCID₅₀/mL. Animal challengestudies showed complete protection from viremia in 1 μg to 40 μg of thedose groups tested. Hence the BPL and formalin inactivated vaccineformulations were further tested at 0.5 μg, and at 0.25 μg 0.125 μg perdose by the IM route in Balb/c mice and were found to be immunogeniceven at low dilutions. For the alum adjuvanted formulations, 0.25 mg ofaluminum (as aluminum hydroxide) per dose was used as the placebocontrol and for non-adjuvanted formulations, 10 mM phosphate buffercontaining 154 mM NaCl, 1% sorbitol and 0.5% L-Glycine, pH 7.40 was usedas the vehicle control. All the formalin and BPL inactivatedformulations elicited high level of neutralizing antibodies andprotected against viremia as depicted in FIG. 5A, FIG. 5B and FIG. 6.Antigen only formulations also elicited high level of neutralizingantibodies and was protected from virus challenge. Recombinant prMEprotein expressed in insect cells was formulated at two doses of 10 and20 μg per dose with 0.25 mg aluminum (as aluminum hydroxide) per dose inBalb/c (8 nos) and injected intramuscularly at day 0 and day 21 elicitedneutralizing antibodies and the data is provided in Table 1. Gammairradiated and Hydrogen peroxide inactivated Zika virus antigen at doseconcentration of 10 μg and formulated with 0.25 mg aluminum (as aluminumhydroxide) per dose was injected by IM route in Balb/c mice at day 0 andday 21 and blood was withdrawn on day 28 for estimation of neutralizingantibodies by PRNT₅₀. Formalin inactivated virus antigen at 10 μg wasformulated with each of the adjuvants disclosed in Example 5 and wasinjected intramuscularly in 4-6 week old Balb/c mice (5 nos per dosegroup) and the blood was drawn at 21 days after vaccine administrationfor estimation of neutralizing antibodies and cytokines. Control groupswas included for each of the adjuvants and no neutralizing antibodies(<10 by PRNT₅₀) could be detected and the data is not shown.Neutralizing antibody titers by PRNT₅₀ of the different adjuvantedformulations, used pooled sera from each group is presented in FIG. 4.High level of neutralizing antibodies were elicited by theaforementioned adjuvanted formulations. It is pertinent to mention thatantibodies to recombinant Zika prME, which is the sequence of the Asiangenotype efficiently cross neutralize the MR766 strain of the Africangenotype. The titers are provided in Table 1.

A combination vaccine of arbovirus antigens were prepared a thefollowing concentrations and tested in Balb/c mice: a) 10 μg formalininactivated Zika virus antigen, 20 μg of BPL inactivated Chikungunyavirus antigen and 6 μg of formalin inactivated JE antigen in a trivalentvaccine combination b) 10 μg formalin inactivated Zika virus antigen and20 μg of BPL inactivated CHIKV virus antigen c) 10 μg of formalininactivated Zika virus antigen and 6 μg of JE virus antigen. Zika viruswas inactivated by 1:2500 of formalin:virus v/v by methods disclosed inExample 3. CHIKV was inactivated by 1:1500 of BPL:virus v/v, and JEV by1:2500 v/v of formalin:virus and tested for completeness of virusinactivation by aforementioned procedures. All the above vaccinecombinations were tested with 0.25 mg aluminum (as aluminum hydroxide)per dose in Balb/c mice (8 nos each) with appropriate controls thatincluded either of the aforementioned antigens alone, and also controlanimals that received equivalent amount of alum. The animals wereboosted at 14 and at 21 days after the first immunization. Blood wascollected at 7 days after the last booster injection. The sera sampleswere used for estimation of neutralizing antibody by PRNT₅₀ for Zika,CHIKV and JEV. The buffer used in all the formulations was 10 mMphosphate buffer, pH 7.2 to 7.6 containing 154 mM NaCl. All the methodsdisclosed above are applicable to any genotype/genotypicvariants/serotypes and strains of Chikungunya virus, Zika virus andJapanese encephalitis viruses. See Table 1 for the results.

TABLE 1 Neutralizing antibodies elicited by various antigenicformulations as disclosed in the Examples. Neutralizing antibody titersas Log10PRNT₅₀ Test Groups Zika A CHIKV JE Recombinant Zika prME - 10 μg× 2.8 — — 2 doses Recombinant Zika prME - 20 μg × 3.22 — — 2 dosesHydrogen peroxide inactivated 2.6 — — Zika antigen - 10 μg × 2 dosesGamma irradiated Zika antigen 2.71 — — 10 μg × 2 doses Zika alumadsorbed - 10 μg × 3.06 — — 3 doses Chikungunya alum adsorbed - —  2.808— 20 μg × 3 doses JE alum adsorbed - 6 μg × — — 3.06 3 doses Zika (10μg) + CHIKV (20 μg) × 2.95 2.68 — 3 doses Zika (10 μg) + JE (6 μg) ×2.80 — 3.28 3 doses Zika (10 μg) + CHIKV (20 μg) + 2.79 2.63 3.21 JE (6μg) × 3 doses

Table 1 Legend:

Purified recombinant prME antigen of Zika virus and the hydrogenperoxide inactivated and gamma irradiated Zika virus antigens formulatedwith 0.25 mg of aluminum per dose elicited neutralizing antibodies inBalb/c mice. The titers are expressed as Log 10PRNT50 values. Thevaccine combinations elicited neutralizing antibodies when two or moreantigens were administered in a single formulation, and no significantantigenic interference was observed between JE, Zika and CHIKV viruses.The titers were estimated in pooled sera samples from each group.

Example 8: Passive Immunization Studies

The proof of concept that neutralizing antibodies are important immunecorrelates of protection against Zika virus infection was demonstratedby single injection of rabbit polyclonal Zika antisera with known titer,about 200 μL of antisera diluted 1:1 with PBS was injectedintraperitoneally in Balb/c mice and challenged 8-24 hours later with10e5 PFU of Zika virus by intravenous route in a volume of 100 μL. Equalno. of control animals received PBS, pH 7.4 and received the virusinjection as the test animals. Blood was collected at 24, 48, 72, 96 and144 hours post virus challenge for detection of viremia in both thegroup of animals. Passive immunization offered complete protectionagainst viremia and infectious virus could not be detected by TCID50.See FIG. 7. Viremia was detected in the control animals that persistedup to 6 days after virus challenge. Zika antibodies could be used as atherapeutic to ameliorate, eradicate or prevent Zika virus infections.

Example 9: Assays for Neutralizing Antibody Titers

Animal sera from all the aforementioned vaccine testing in micedescribed in Example 7 which include all the monovalent Zika vaccinesinactivated with different inactivating agents and formulated withdifferent adjuvants, vaccine antisera from dose ranging studies as wellcombination vaccines with CHIKV and JEV described in the precedingsections were assayed for neutralizing antibodies by 50% PlaqueReduction Neutralization Test (PRNT₅₀) by standardized procedures.Briefly, one day prior to the assay, 6-well plates were seeded with2.5×10³ Vero cells (ATCC CCL-81) per well and the plates were incubatedat 37° C. in a 5% CO₂ incubator. To 4-fold dilutions of the sera samplesin MEM containing equal volume of the standardized Zika virus strain(10⁵ pfu/mL) was added and incubated at 37° C. with 5% CO₂ for 90 min.The cells were washed twice with 1×PBS pH 7.4 (10 mM phosphate with 150mM NaCl) and 0.30 ml of each dilution of the serum-virus mixture wasadded to the corresponding well and incubated for 90 min at 37° C. in a5% CO₂ incubator. Each assay was carried out in triplicates. The cellswere overlaid with 2 ml of 0.85% methyl cellulose in MEM with 1%penicillin-streptomycin and 1% L-glutamine. The plates were incubated at37° C. in a 5% CO₂ incubator for 4 days. At the end of incubation, theplaques were fixed with 10% formalin, washed with 1×PBS, pH 7.4 and werevisualized with 0.1% crystal violet. The highest dilution of serumcausing 50% reduction in the number of plaques formed by the controlvirus sample was estimated as the PRNT₅₀ titer. Anti-CHIKV and anti-JEantibodies from the vaccine combinations were also estimated PRNT50. Allthe aforementioned vaccine antigens elicited high level of neutralizingantibodies as depicted in FIGS. 5 and 6

Example 10: Zika Virus Cross Neutralization Studies

Formalin inactivated vaccine antisera cross neutralized the homologousMR766 virus strain of the African genotype and FSS13025 Zika virusstrain (GenBank Acc No. JN860885) of the Asian genotype with EQUALefficiency with PRNT50 titers of 18105 and 18325 against MR766 andFS13025 strains respectively. (The study BS-3018 was contracted to IBTBioservices, Gaithersburg, Md., USA). Briefly, both the MR766 and theFS13025 Zika virus strains were diluted to ˜250 PFU in serum-freemedium. Both the vaccine antisera and control sera (placebo) wereserially diluted in two-fold dilutions. The virus samples were mixed 1:1with serially diluted sera samples and incubated at 37° C. for 2 hours.Vero cells seeded in 24-well plates were infected with the dilutions for1 hour and 0.85% methyl cellulose was added to each well and incubatedfor 3 days. Cells were fixed and analyzed by plaque assay. The plateswere scanned and the plaque counts were used to calculate the PRNT₅₀titers using a 4PL curve fit. Hence the method of vaccine antigenpreparation, formulation and testing are entirely applicable across anygenotype of Zika virus as the vaccine with one genotype 100% crossneutralizes the heterologous strain and this also proves that noserotypes of Zika virus exists and that inactivated vaccine of Zikausing any strain will be equally protective and potent as vaccineprepared using any genotype, and genotypic variant or indeed any Zikavirus strain. This fact was further corroborated when the antibodiesraised against the recombinant protein expressed as prME (protein of SEQID NO:3) in insect cells cross neutralized the MR766 virus with highefficiency. The protein of SEQ ID No. 3 is derived from the prMEsequence of the Zika virus strain H/PF/013, which is the morecontemporary strain of the Asian genotype. Cross neutralization of thevaccine antisera of the homologous MR766 strain of nucleotide SEQ IDNO:5 encoding the complete ORF of SEQ ID NO:6 and the heterologousFSS13025 of the SEQ ID NO:7 encoding the complete ORF of SEQ ID NO: 8 isdepicted in FIG. 8A and FIG. 8B.

Example 11: Antibody ELISA

Briefly, Zika virus antigen was coated at the standardized concentrationin coating buffer in 96-well plates overnight at 2 to 8° C. The platecontents were discarded and the wells were blocked with blocking bufferand washed extensively before adding the vaccine antisera at serialdilutions. Each vaccine antisera was assayed in triplicates. The plateswere incubated for 90 min at 37° C., before adding secondary antibody(anti mouse-IgG HRPO conjugate) diluted 1:2500 in antibody diluentbuffer. Each of the wells were washed five times with washing buffer(PBST, pH 7.4) and three times with PBS (pH 7.4), 30 seconds each. About100 μl/well of freshly prepared substrate solution was added andincubated at ambient temperature for 10 minutes for color development.The color development was stopped by addition of 50 μL/well stopsolution. Absorbance was read at 492 nm and the results recorded. Foreach assay, antigen blank, primary and secondary antibody blanks wereincluded as controls. Seroconversion cut off value=pre-exposure averagetiter+(3× standard deviation). The end point dilution of positivelyseroconverted sample which shows a titer equivalent to the pre-exposurelevel titer was identified. Reciprocal of the penultimate dilution ofend point of a positively seroconverted sample was interpreted as theantibody endpoint titer. Antibody titers to both BPL inactivated andformalin inactivated Zika vaccine formulations were higher with aluminumhydroxide than with antigens alone. Vaccine formulations of the formalininactivated vaccine at all doses (FIG. 9A-9C) and all doses of BPLinactivated vaccine (data not shown) elicited high level of antibodies,after each dose of vaccine administration confirming that vaccine can beadministered as a single dose or two or more doses for eliciting arobust immune response against Zika virus.

Example 12: Antibody Avidity

The quality of antibody responses to the vaccine was estimated byantibody avidity assays. The antigen-antibody binding avidities are thedegree of affinity maturation in the B-cells. Higher antibody aviditiescorrelate with neutralizing antibodies in several vaccine studies. Priorto determination of avidity index, titrations with sodium isothiocyanate(NaSCN) from 0 M to 6 M concentration in 0.25 M steps from 0 to 2.0 Mwere performed. After addition and incubation of primary antisera to theantigen coated plates, the plates were incubated with gradedconcentrations NaSCN for 15 min with intermittent shaking, washed anddeveloped as in regular ELISA. The optical densities obtained at each ofthe concentrations were plotted. The highest OD (A) was plotted andhalved (A/2), and the distance between the OD curves at A/2 was measuredas the NaSCN shift value. The NaSCN shift was higher after the firstbooster dose compared to the prime dose and remained static ormarginally increased further after second booster dose administrationindicating that high affinity antibodies developed over time and withbooster injections. A reference point in the ELISA titration was takencalculation of avidity index, (AI]) which is the ratio of antibodyconcentration (measured by absorbance) in ELISA of serum samples treatedwith and without the chaotropic agent NaSCN. Even at the lowest singledose concentration of 1 μg of formalin inactivated Zika vaccine,antibodies with high affinity binding to the antigen was detected,indicating the vaccine is potent even at low concentrations of thevaccine antigen (See FIG. 10).

Example 13: Cytokine Profiling

Both Th1 and Th2 cytokines were estimated in mice sera afteradministration of two doses of the formalin inactivated Zika antigenformulated with different adjuvants including aluminium hydroxide and inantigen only controls for comparison. The Mouse ELISA kit—Th1/Th2(Catalog No. 88-7711-44, eBioscience) was used for the estimation ofIL-2, IFN gamma, IL-4 and IL-10 by methods exactly as per the kitprotocols using the standards provided in the kit. The concentration ofthe cytokines are expressed in μg/mL. The results for Th1 cytokinelevels are depicted in FIG. 11A and FIG. 11B and Th2 cytokines in FIG.12A and FIG. 12B.

Example 14: Estimation of Virus Titers

The amount of infectious virus particles in the upstream and downstreambioprocess samples, Zika virus titers for animal challenge studies weremeasured by TCID50 (50% Tissue Culture Infectious Dose) assay. Thisassay measures the dilution of the virus sample that generatescytopathic effect (CPE) in 50% of the cells. Vero cells were seeded in96-well microplates and incubated in 5% CO₂ at 37° C. overnight. Thecells were infected with 10-fold serial dilutions of virus sample,followed by incubation for 5 d in 5% CO₂ at 33° C. The cells werevisually inspected for CPE and the TCID50 titer was calculated accordingto the method of Reed and Muench, 1938. The results are presented as alog 10 titer (10×TCID50 units/mL). Alternatively plaque assays were usedand the titers were expressed as plaque forming units, PFU/mL. Theprotocol is similar to PRNT50 assays described in Example 9 except thatthe incubation of the virus with serially diluted sera samples is notcarried out.

REFERENCES

-   1. Cooper P D, Steele E J. The adjuvanticity of gamma inulin.    Immunol Cell Biol. 1988, 66:345-52.-   2. Reed L J. Muench H. A simple method of estimating fifty percent    endpoints. Am. J. Epidemiol. (1938) 27 (3): 493-497    4849-0458-9365, v. 1

We claim:
 1. An immunogenic composition comprising a structural antigenof Zika virus and a structural antigen of Chikungunya virus, wherein thestructural antigen of Zika virus comprises an amino acid sequenceencoded by the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or the aminoacid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4; and apharmaceutically acceptable buffer; wherein the Zika virus and theChikungunya virus are subjected to inactivation such that thecomposition is rendered non-infectious, and wherein the compositionelicits an immune response to Zika virus and Chikungunya virus inmammals.
 2. The composition according to claim 1, wherein the Zika virusand/or the Chikungunya virus is a whole virus.
 3. The compositionaccording to claim 2, wherein the inactivation was performed by exposureof the whole virus to one or more treatments selected from the groupconsisting of a treatment with a chemical inactivating agent, treatmentwith a physical inactivating agent, treatments with an irradiatingagent, and heat treatments.
 4. The composition according to claim 3,wherein the chemical inactivating agent is selected from the groupconsisting of formalin (formaldehyde), beta propiolactone (BPL) andhydrogen peroxide.
 5. The composition according to claim 3, wherein theirradiating agent comprises gamma irradiation or UV irradiation.
 6. Thecomposition according to claim 1, wherein the Zika virus and/or theChikungunya virus is purified and inactivation is carried out before orafter purification.
 7. The composition according to claim 1, wherein theZika antigen comprises one or more antigens selected from envelope (E)protein, membrane (M) protein, and prME protein, obtained or derivedfrom the Zika virus.
 8. The composition according to claim 1, whereinthe Chikungunya antigen comprises one or more antigens selected from thegroup consisting of capsid protein, E1 protein, E2 protein, and 6kprotein, obtained or derived from the Chikungunya virus.
 9. Thecomposition according to claim 1, wherein the Zika antigen and/or theChikungunya antigen are expressed as virus like particles in aprokaryotic or eukaryotic expression system.
 10. The compositionaccording to claim 1, wherein the composition elicits Th1 and/or Th2immune response to infection by Zika virus and Chikungunya virus. 11.The composition according to claim 1, wherein the buffer is selectedfrom the group consisting of a phosphate buffer, a sodium chloridebuffer, a citrate buffer, a phosphate citrate buffer, a borate buffer, atris(hydroxymethly)aminomethane (Tris) containing buffer, a succinatebuffer, and buffers containing glycine or histidine as one of thebuffering agents.
 12. The composition according to claim 11, wherein thephosphate buffer comprises sodium phosphate at concentration of 5 mM upto 200 mM of phosphate ions at a pH of 6.5 to 9, or the sodium chloridebuffer comprises sodium chloride at a concentration of 50 mM to 200 mM.13. The composition according to claim 1, wherein the compositionfurther comprises an adjuvant.
 14. The composition according to claim13, wherein the adjuvant is selected from the group consisting of: analuminum salt; inulin; algammaulin; monophosphoryl lipid A (MPL);resiquimod; muramyl dipeptide (MDP); N-glycolyl dipeptide (GMDP);polylC; CpG oligonucleotide; aluminum hydroxide with MPL; water in oilemulsion; oil in water emulsion; and combinations thereof.
 15. Thecomposition according to claim 14, wherein the composition comprisesaluminum hydroxide at a concentration of from 0.1 mg to 1.5 mg ofaluminum per vaccine dose.
 16. The composition according to claim 1,which comprises 2-phenoxyethanol at a concentration of 2.5 to 5 mg/mL.17. The composition according to claim 1, wherein the composition is ina liquid or a lyophilized form.
 18. The composition according to claim1, wherein the composition is contained within pre-filled syringes,microneedle patch, needle-free patch, and/or inhalation or nasal sprays.19. An Immunogenic composition comprising: one or more recombinantantigens of a structural protein of a Zika virus and one or morerecombinant antigens of a structural protein nucleic acid of aChikungunya virus, and a pharmaceutically acceptable buffer, wherein thestructural protein of Zika virus comprises an amino acid sequenceencoded by the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or the aminoacid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4; and whereinthe composition elicits an immune response to infection by Zika virusand Chikungunya virus in a mammal.
 20. The composition according toclaim 19, wherein the one or more recombinant antigens are selected fromthe envelope (E) protein, membrane (M) protein, and prME protein,obtained or derived from the Zika virus, and one or more recombinantantigens are selected from capsid protein, E1 protein, E2 protein, and6k protein, obtained or derived from the Chikungunya virus.
 21. Thecomposition according to claim 19, wherein the one or more recombinantantigens are expressed as virus like particles in a prokaryotic oreukaryotic expression system.
 22. The composition according to claim 19,which elicits Th1 and/or Th2 immune response against infection by Zikavirus and by Chikungunya virus in the mammal.
 23. The compositionaccording to claim 19, wherein the buffer is selected from the groupconsisting of a phosphate buffer, a sodium chloride buffer, a citratebuffer, a phosphate citrate buffer, a borate buffer, atris(hydroxymethly)aminomethane (Tris) containing buffer, a succinatebuffer, and buffers containing glycine or histidine as one of thebuffering agents.
 24. The composition according to claim 23, wherein thephosphate buffer comprises sodium phosphate at concentration of 5 mM upto 200 mM of phosphate ions at a pH of 6.5 to 9 or the sodium chloridebuffer comprises sodium chloride at a concentration of 50 mM to 200 mM.25. The composition according to claim 19, wherein the compositioncomprises an adjuvant.
 26. The composition according to claim 25,wherein the adjuvant is selected from the group consisting of analuminum, inulin, algammaulin, monophosphoryl lipid A (MPL), resiquimod,muramyl dipeptide (MDP), N-glycolyl dipeptide (GMDP), polylC, CpGoligonucleotide, aluminum hydroxide with MPL, water in oil emulsion, oilin water emulsion, and combinations thereof.
 27. The compositionaccording to claim 26, wherein the adjuvant comprises aluminum hydroxideat a concentration of 0.1 mg to 1.5 mg of aluminum per vaccine dose. 28.The composition according to claim 19, wherein the composition comprises2-phenoxyethanol at a concentration of 2.5 to 5 mg/mL.
 29. Thecomposition according to claim 19, which is aqueous or lyophilized. 30.The composition according to claim 19, which is contained withinpre-filled syringes, microneedle patch, needle-free patch, and/orinhalation or nasal sprays.
 31. The composition of claim 4, wherein theinactivation comprises a treatment selected from the group consistingof: (a) treatment with formalin at a formalin to virus ratio of 1:500 to1:4000 (v/v) at 8° C. to 37° C. for 1 to 7 days; (b) treatment withformalin at a formalin to virus ratio of 1:500 to 1:4000 (v/v) at 2° C.to 8° C. for 10 to 30 days; (c) treatment with formalin at a formalin tovirus ratio of 1:500 to 1:4000 (v/v) at 25±3° C. for 1 to 7 days; (d)treatment with Beta-propiolactone (BPL) at a BPL to virus ratio of 1:500to 1:4000 (v/v) at 8° C. to 30° C. for 24 to 48 hours; (e) treatmentwith BPL at a BPL to virus ratio of 1:500 to 1:4000 (v/v) at 2° C. to 8°C. for 3 to 7 days; (f) treatment with BPL at a BPL to virus ratio of1:500 to 1:4000 (v/v) at 25±3° C. for 48 hours; (g) treatment withformalin as set forth in a, b, or c, plus treatment with BPL as setforth in d, e, or f; (h) treatment with formalin at a formalin to virusratio of 1:3000 (v/v) at 15° C. to 30° C. for 24 to 48 hours plustreatment with BPL and BPL to virus ration of 1:3000 (v/v) at 15° C. to30° C. for 24 hours; (i) treatment with formalin at a formalin to virusratio of 1:3000 at 25±3° C. for 24 to 48 hours plus treatment with BPLat a BPL to virus ration of 1:3000 (v/v) at 25±3° C. for 24 hours; (j)treatment with hydrogen peroxide at a hydrogen peroxide concentration of0.1% to 3% at 20° C. to 30° C. for 5 minutes to 120 minutes; and (k)treatment with hydrogen peroxide at a hydrogen peroxide concentration of0.1% to 1% at 20° C. to 30° C. for 5 minutes to 120 minutes.
 32. Animmunogenic composition comprising an antigen obtained or derived from aZika Virus, and a pharmaceutically acceptable buffer, wherein theantigen is rendered non infectious through inactivation and/or isrecombinantly engineered from the nucleic acid of a Zika virus; and theantigen contains the amino acid sequence of SEQ ID NO 3 and/or SEQ ID NO4.
 33. The immunogenic composition of claim 32, wherein the Zika virusand the CHIKV virus are inactivated and inactivation comprises atreatment selected from the group consisting of: (a) treatment withformalin at a formalin to virus ratio of 1:500 to 1:4000 (v/v) at 8° C.to 37° C. for 1 to 7 days; (b) treatment with formalin at a formalin tovirus ratio of 1:500 to 1:4000 (v/v) at 2° C. to 8° C. for 10 to 30days; (c) treatment with formalin at a formalin to virus ratio of 1:500to 1:4000 (v/v) at 25±3° C. for 1 to 7 days; (d) treatment withBeta-propiolactone (BPL) at a BPL to virus ratio of 1:500 to 1:4000(v/v) at 8° C. to 30° C. for 24 to 48 hours; (e) treatment with BPL at aBPL to virus ratio of 1:500 to 1:4000 (v/v) at 2° C. to 8° C. for 3 to 7days; (f) treatment with BPL at a BPL to virus ratio of 1:500 to 1:4000(v/v) at 25±3° C. for 48 hours; (g) treatment with formalin as set forthin a, b, or c, plus treatment with BPL as set forth in d, e, or f; (h)treatment with formalin at a formalin to virus ratio of 1:3000 (v/v) at15° C. to 30° C. for 24 to 48 hours plus treatment with BPL and BPL tovirus ration of 1:3000 (v/v) at 15° C. to 30° C. for 24 hours; (i)treatment with formalin at a formalin to virus ratio of 1:3000 at 25±3°C. for 24 to 48 hours plus treatment with BPL at a BPL to virus rationof 1:3000 (v/v) at 25±3° C. for 24 hours; (j) treatment with hydrogenperoxide at a hydrogen peroxide concentration of 0.1% to 3% at 20° C. to30° C. for 5 minutes to 120 minutes; and (k) treatment with hydrogenperoxide at a hydrogen peroxide concentration of 0.1% to 1% at 20° C. to30° C. for 5 minutes to 120 minutes.
 34. The composition of claim 14,wherein the aluminum salt comprises aluminum hydroxide, aluminumphosphate or aluminum sulfate phosphate.
 35. The composition of claim26, wherein the aluminum salt comprises aluminum hydroxide, aluminumphosphate or aluminum sulfate phosphate.